Although the free concentration is more significant in the environmental chemistry and toxicology of receptor-mediated toxicants, few studies have been conducted to use it as a dose-metric. The relative binding affinity of three model endocrine disrupting compounds, diethylstilbestrol (DES), ethynylestradiol (EE2), and bisphenol A (BPA), were evaluated using a competitive ELISA with human estrogen receptor alpha. After measuring the available receptors and the dissociation constant for 17beta-estradiol, binding inhibition curves using the free concentration as the dose-metric were obtained by assuming species equilibrium in the ELISA system and compared with apparent inhibition curves generated using the nominal concentration as the dose-metric. Because ligand binding to estrogen receptors may reduce its free concentration in the assay system, the differences between the two curves for free and nominal concentrations are more significant for more strongly binding ligands. The ratio of a compound's nominal concentration causing 50% inhibition (IC50) to the IC50 of DES, the positive control, was strongly affected by specific assay conditions, while that estimated by modeling free concentration is independent of receptor concentration, indicating that the free concentration is a better dose-metric for a competitive binding assay.