Using the reaction rate of p-nitrophenyl acetate (NPA) with human serum albumin (HSA) as an index, the inhibition modes and binding sites of various anionic drugs were investigated in pH 7.4 phosphate buffer at 25°. Eleven anionic drugs including two specific fluorescence probes were chosen on the basis of Sudlow's classification of the drug binding sites, Site I and Site II (Sudlow et al., Mol. Pharmacol., 12, 1052 (1976)). The types of inhibition caused by these drugs suggested that at least three binding sites were present on HSA. The primarily reactive site (R site) of HSA corresponded to Sudlow's Site II, which corresponds to tyrosine-411 in Brown's HSA sequence. Site I was not involved in the esterase activity of HSA, which required tryptophan-214 (U site). In addition to the R site, HSA had a secondarily reactive site (T site) for NPA, which could also bind the drugs. The binding affinities of the drugs to the R site were estimated and some structural features of the R site were deduced.