Twenty-seven substituted benzoic acids have been studied as inhibitors of partially purified human renal thiopurine methyltransferase (TPMT). Quantitative structure-activity relationship (QSAR) analysis resulted in the following equation: pI50 = 1.25( +/- 0.53)pi'3 + 0.73( +/- 0.38)MR3,4 + 2.92( +/- 0.39). In this equation pI50 is the -log of the concentration of compound that inhibits the enzyme activity by 50% (IC50);pi'3 is the relative hydrophobicity of the more hydrophobic of the two meta substituents; and MR3,4 is the molar refractivity of the more hydrophobic of the two meta substituents and of the para substituent on the phenyl ring. In addition, 14 substituted thiophenols were tested as substrates for the enzyme. All 14 thiophenols tested were excellent substrates with Km constants (0.8-7.8 microM) that were at least 2 orders of magnitude lower than those of any known thiopurine substrate for TPMT. However, there was no discernible relationship between the activities of thiophenol substrates and their physicochemical parameters. These results suggest that benzoic acid inhibitors of and thiophenol substrates for TPMT may interact with different sites on the enzyme.