Using selective cytochrome P450 (CYP) inhibitors and clinical concentrations (4 microM) of dapsone (DDS), we found a major contribution of CYP2C9 and little or no contribution (< or = 10%) of CYP3A4 and CYP2E1 to dapsone N-hydroxylation (DDS-NHY) in human liver microsomes. Sulfaphenazole (2.16 microM) and tolbutamide (500 microM), selective inhibitors of CYP2C9 (or 2C8/9), inhibited DDS-NHY by 48 +/- 14 and 41 +/- 15%, respectively. The apparent Michaelis-Menten Km values for DDS-NHY by cloned CYP2C8, CYP2C9, CYP2C18, and CYP2C19 were 75 microM, 31 microM, 25 microM, and greater than 1 mM, respectively. CYP3A4 and CYP2E1 were incapable of DDS-NHY at 4 microM DDS. S-mephenytoin (360 microM) activated DDS-NHY by human liver microsomes and by CYP2C8 by 43 +/- 36 and 193 +/- 16%, respectively. This activation was cytochrome b5-dependent. In contrast, S-mephenytoin inhibited DDS-NHY by CYP2C9, CYP2C18, and CYP2C19 by 27 +/- 2, 49 +/- 1, and 32 +/- 4%, respectively. Because CYP2C18 and CYP19 are expressed at low concentrations in the human liver, these observations indicate that at clinical DDS concentrations, CYP2C9 is a major and CYP2C8 is a likely minor contributor to DDS-NHY in human liver microsomes.