Methoxsalen (8-methoxypsoralen) is a very potent inhibitor of human cytochrome P450 2A6 (CYP2A6) and mouse Cyp2a-5-mediated coumarin 7-hydroxylation in vitro. To determine the effect of methoxsalen on coumarin 7-hydroxylation in humans in vivo, five subjects were given 45 mg of methoxsalen and 5 mg of coumarin. Methoxsalen inhibited in vivo coumarin metabolism by 47 +/- 9.2% (mean +/- SEM). Methoxsalen was metabolized in human liver microsomes at the rate of 50-100 pmol/mg protein/min (approx. 30% of the activity in mouse liver microsomes). Metabolism was not inhibited by the anti-Cyp2a-5 antibody in human liver microsomes. NIH 3T3 cells stably expressing catalytically active CYP2A6 enzyme did not metabolize methoxsalen, indicating that CYP2A6 does not accept methoxsalen as a substrate. In pyrazole-induced mouse liver microsomes, methoxsalen metabolism was inhibited by the anti-Cyp2a-5 antibody. Cyp2a-5 protein expressed in the yeast Saccharomyces cerevisiae was capable of metabolizing methoxsalen, indicating that methoxsalen is a substrate of Cyp2a-5. Although kinetic studies indicated that the inhibition of coumarin 7-hydroxylation by methoxsalen is competitive in human liver microsomes, methoxsalen does not appear to be a substrate for CYP2A6. Methoxsalen and coumarin have the potential of strong metabolic interactions in man.