Effects of ligand depletion on heterologous competition data depend on the relative affinity of the inhibitor. A-F. Competition data for [3H]EB and nicotine were generated with two sites modelfree with [3H]EB = 0.013 (A & B), 0.3 (C & D), and 20 nM (E & F) and the R1T values shown in C. The y-axes of A, C, and E show total bound [3H]EB; y-axes of B, D, and F show normalized binding for comparing IC50 values. Data from small values of R1T are not distinguishable because of the ranges of the y-axis scales (A, C, and E) or because data sets overlap when rightward shifts of IC50 are negligible (B, D, and F). Ligand depletion shifts IC50 rightward; shape of the competition curve remains approximately sigmoidal. G-L. Competition data for [3H]EB and a hypothetical superhigh affinity competitor were generated with two sites modelfree with [3H]EB = 0.013 (G & H), 0.3 (I & J), and 20 nM (K & L) and the R1T values shown in L. The two inhibition constants Ki1 and Ki2 were 100-fold tighter (1.3 × 10-4 and 0.12 nM) than Kd1 and Kd2 for [3H]EB. Ligand depletion shifts IC50 rightward and increases the maximum steepness of the negative slope of the sigmoidal shape. Hill coefficients at R1T = 130 nM are -35, -35, and -17 for [3H]EB = 0.013, 0.3, and 20 nM.