Granisetron, a potent 5-HT3 receptor antagonist, has been reported to be mainly metabolized to 7- hydroxygranisetron and a lesser extent to 9-desmethylgranisetron in humans. A previous study indicated that cytochrome P450 (CYP)3A4 is a major catalyst of 9-demethylation, although the major CYP isoform(s) responsible for 7- hydroxylation are unknown. To clarify granisetron 7-hydroxylase, the in vitro metabolism of granisetron using expressed human CYPs and human liver microsomes was investigated. 7-Hydroxygranisetron was produced almost exclusively by CYP1A1, while, apparently, 9-desmethylgranisetron was preferentially produced by CYP3A4. Marked inter-individual differences in the ratio of the formation of 7-hydroxygranisetron and 9-desmethylgranisetron in human liver microsomes was observed. Granisetron 7-hydroxylase activity was strongly correlated with benzo[a]pyrene 3-hydroxylase activity (p < 0.0001), but not with testosterone 6b-hydroxylase activity in human liver microsomes. Furthermore, an anti-human CYP1A1 antibody completely inhibited 7-hydroxylation in human liver microsomes, however, the reaction was not inhibited at all by an anti-CYP3A4 antibody. On the other hand, granisetron 9-demethylase activity correlated significantly not only with testosterone 6b-hydroxylase activity (p < 0.0001) but also with benzo[a]pyrene 3-hydroxylase activity (p < 0.01). Consistent with this, both the anti-CYP1A1 and anti-human CYP3A4 antibodies inhibited the 9-demethylase activity. These data indicate that CYP1A1 is a major enzyme responsible for the metabolism of granisetron via a main 7- hydroxylation pathway and an alternative 9-demethylation route. This is the first report demonstrating the substantial contribution of CYP1A1 to the metabolism of a drug, although its role in the metabolism of environmental compounds is well established.