We studied the effects of acetonitrile, dimethyl sulfoxide (DMSO), and methanol (MeOH) in human hepatocytes on cytochrome P450 (CYP) and phase II conjugation activities: phenacetin O-deethylation (CYP1A2), coumarin 7-hydroxylation (CYP2A6), tolbutamide 4-hydroxylation (CYP2C9), S-mephenytoin 4'-hydroxylation (CYP2C19), dextromethorphan O-demethylation (CYP2D6), chlorzoxazone 6-hydroxylation (CYP2E1), testosterone 6beta-hydroxylation (CYP3A4), and umbelliferone glucuronidation and sulfation. The solvents were evaluated at concentrations (v/v) of 0.1, 1, and 2%. Previously cryopreserved human hepatocytes pooled from multiple donors were used as suspension cultures in this study. DMSO was found to inhibit CYP2C9 and CYP2C19, CYP2E1, and CYP3A4 in a concentration-dependent manner. At 2% DMSO, the activities for the four isoforms were approximately 40% (CYP2C9), 23% (CYP2C19), and 11% (CYP2E1) of that observed for 0.1% acetonitrile and 45% (CYP3A4) of that observed for 1% acetonitrile. No apparent inhibitory effects were observed for the other activities evaluated. Methanol was found to inhibit CYP2C9 and CYP2E1 activities, but to a lesser extent than DMSO. Acetonitrile had no apparent effects on any of the on any of the activities evaluated. These findings should be considered when choosing an organic solvent for metabolism studies with human hepatocytes.