Species differences in microsomal binding were evaluated for 43 drug molecules in human, monkey, dog and rat liver microsomes, using a fixed concentration of microsomal protein. The dataset included 32 named drugs and 11 proprietary compounds encompassing a broad spectrum of physicochemical properties (11 acids, 24 bases, 8 neutral, c log D -1 to 7, MW 200 to 700 and free fraction <0.001 to 1). Free fractions (f(u,mic)) in monkey, dog, rat and human microsomes were highly correlated, with linear regression correlation coefficients greater than 0.97. The average fold-difference in f(u,mic) between monkey, dog, or rat, and human was 1.6-, 1.3-, and 1.5-fold, respectively. Species differences in f(u,mic) were also assessed for a range of microsomal protein concentrations (0.2-2 mg/mL) for midazolam, clomipramine, astemizole, and tamoxifen, drugs with low to high microsomal binding. The mean fold species-difference in f(u,mic) for midazolam, clomipramine, astemizole, and tamoxifen was 1.1-, 1.2-, 1.3-, and 2.0-fold, respectively, and was independent of normalized microsomal protein concentration. For a fixed concentration of microsomal protein, greater than 76% and 90% of drugs examined in this study had preclinical species f(u,mic) within 1.5- and 2-fold, respectively, of experimentally measured human values.