Human liver microsomes had about 8-fold higher 7-hydroxylation activities for S-warfarin than for R-warfarin. Activities of racemic warfarin 7-hydroxylation by liver microsomes of 35 human samples correlated more closely with those of S-warfarin 7-hydroxylation (r = 0.95) than with those of R-warfarin 7-hydroxylation (r = 0.69). The correlation coefficient between R-warfarin 7-hydroxylation and 7-ethoxyresorufin O-deethylation activities was 0.73 in these human samples, suggesting that R- and S-warfarin enantiomers are catalyzed by different forms of human cytochrome P450 (P450 or CYP) enzymes. Anti-CYP2C9 antibodies inhibited completely the 7-hydroxylation of S-warfarin, but not R-warfarin, catalyzed by human liver microsomes, while anti-CYP1A2 inhibited R-warfarin 7-hydroxylation by about 70%. Interestingly, the racemic warfarin 7-hydroxylation activities (turnover numbers of 1.6 +/- 1.0 pmol/min/mg protein in 35 human samples) were found to be low compared with the S-warfarin 7-hydroxylation activities (4.1 +/- 2.5 pmol/min/mg protein), indicating that R-warfarin may have affected the CYP2C9-dependent S-warfarin 7-hydroxylation activities when racemic warfarin was used as a substrate. Several P450 inhibitors, as well as R-warfarin, were examined for their abilities to inhibit S-warfarin 7-hydroxylation; we found that R-warfarin was a non-competitive inhibitor with a Ki value of about 150 microM, whereas both tolbutamide and sulfaphenazole were competitive inhibitors with Ki values of about 100 and 0.5 microM, respectively, for S-warfarin 7-hydroxylation activities. These results suggest that R- and S-warfarin enantiomers are catalyzed principally by CYP1A2 and CYP2C9, respectively, in human liver microsomes, and that the pharmacokinetic properties of S-warfarin may be altered by R-warfarin in vivo when racemic warfarin is administered clinically to humans.