IC50 and EC50 are two of the most reported numbers in pharmacology, toxicology, and bioassay screening — and two of the most frequently misused. They look almost identical on a curve, they share the same fitting machinery, and most software prints them with the same units. But they answer different questions, and reporting one when you mean the other quietly invalidates downstream comparisons.

This post defines both metrics, shows where they come from on a dose–response curve, walks through how they're actually calculated, and lists the small set of decisions that determine whether your IC50/EC50 is reproducible.

Definitions

EC50 — the Effective Concentration that produces 50 % of the maximal response. EC50 is the natural metric for stimulatory assays (agonists, growth, enzyme activation, fluorescence increase): higher signal = more effect.
IC50 — the Inhibitory Concentration that produces 50 % inhibition of a baseline activity. IC50 is the natural metric for inhibitory assays (antagonists, cytotoxicity, enzyme inhibition, viability decrease): higher dose = lower signal.

Both are read off the same 4PL/5PL curve at the same vertical position — the midpoint between the fitted top and bottom asymptotes. The difference is the direction of the response and what "50 %" refers to.

When to report which

Assay type	Response direction	Report
Agonist binding, receptor activation	Signal ↑ with dose	EC50
Cell proliferation, growth factor	Signal ↑ with dose	EC50
Enzyme activator, reporter induction	Signal ↑ with dose	EC50
Cytotoxicity (MTT, CellTiter-Glo)	Signal ↓ with dose	IC50
Enzyme inhibitor, kinase screen	Signal ↓ with dose	IC50
Antimicrobial, antiviral activity	Signal ↓ with dose	IC50 (often as MIC/EC50 too)

If your assay can go in either direction (some functional assays do), pick once per project and stick with it — mixing EC50 and IC50 across plates of the same compound makes potency comparisons meaningless.

How IC50/EC50 is actually calculated

Both come from a non-linear least-squares fit, almost always a 4-parameter logistic (4PL):

y = D + (A − D) / (1 + (x / C)^B)

where:

A = bottom asymptote (response at concentration 0),
D = top asymptote (response at saturating concentration),
C = the inflection point on the dose axis — this is your EC50 / IC50,
B = Hill slope (steepness of the transition).

Asymmetric curves (cooperative binding, biased agonism) need a 5PL with a fifth parameter E that lets the upper and lower halves of the curve have different curvature. A 5PL almost always reports a slightly different EC50 than a 4PL on the same data — usually closer to the true midpoint when the curve is asymmetric.

On a normalised curve (0 % to 100 % response), IC50 and EC50 are exactly the C parameter. On raw signal, they are the concentration where y = (A + D) / 2.

The five decisions that determine reproducibility

An EC50 reported without these five choices is not reproducible:

Model — 4PL or 5PL. Use an F-test, not your eye, to decide whether the extra 5PL parameter is justified.
Normalisation — % of control, fold-change, or raw signal. Different choices give different EC50 values on the same data.
Constraints — were Top and Bottom fixed to control means, or left free? A free fit on a curve that doesn't reach the plateau will drift the asymptote and shift the midpoint.
Weighting — equal weights, 1/Y, or 1/Y². Dose-response data is heteroscedastic; unweighted fits bias the midpoint toward the noisier end of the curve.
Reported confidence interval — a point estimate of EC50 without a 95 % CI is half a result. Two compounds with overlapping CIs are not significantly different in potency, no matter what the point estimates say.

Common pitfalls

Reporting EC50 when the top plateau wasn't reached. If the curve hasn't levelled off, the fit will extrapolate the asymptote and the EC50 will move with every added or removed top point. Either extend the concentration range or constrain Top to a defensible value.

Comparing IC50 across assays. An IC50 from an enzyme assay and an IC50 from a cell viability assay are not the same quantity. Compare within an assay format.

Treating IC50 as affinity. For competitive binding assays, the Cheng–Prusoff equation converts IC50 to the true binding constant Ki, accounting for substrate concentration. Reporting IC50 as Ki is a common error in early SAR work.

Confusing IC50 with potency rank in noisy assays. A 2-fold IC50 difference inside the noise of the assay is not a real potency difference. Always look at the 95 % CI before ranking compounds.

Linear interpolation between two points around 50 %. This was acceptable when curve fitting was hard. It isn't anymore — a full 4PL/5PL fit with proper weighting takes seconds and gives you a CI.

Practical workflow

Plot the data on a log-concentration axis before fitting — your eye is the fastest sanity check for whether a 4PL is even appropriate.
Fit a 4PL with 1/Y weighting; let the engine multi-start to avoid local minima.
Run a 5PL on the same data; compare with an F-test at α = 0.05. Keep the simpler model unless 5PL is significantly better.
Inspect the 95 % CI on EC50. If it spans more than ~3-fold, the assay or the concentration range needs work before the number is publishable.
Report: model, normalisation, constraints, weighting, EC50/IC50 with 95 % CI, Hill slope, R², Z' factor.

In Labreador

The Dose-Response module does all five of the decisions above explicitly. Auto-fit runs the 4PL vs 5PL F-test, normalisation and constraints are first-class options, weighting is selectable per fit, and every EC50/IC50 is reported with a 95 % confidence interval computed from the log-transformed standard error — not a symmetric ± value that crosses zero on potent compounds.

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IC50 vs EC50: What They Mean, How to Calculate Them, and When Each Applies