The interaction of antihistaminics, including mepyramine, with rat hepatic cytochrome P450s (P450s) was investigated. We first investigated mepyramine binding to eight forms of rat hepatic P450s. Mepyramine bound specifically to P450 2D1, which suggests that it inhibits P450 2D activity. Histamine H1 receptor antagonists (mepyramine, diphenhydramine, chlorpheniramine and triprolidine) inhibited the lidocaine 3-hydroxylation activity catalyzed by P450 2D1 but did not inhibit the testosterone hydroxylation activities catalyzed by P450s other than P450 2D forms. The Ki values of these antagonists for the catalytic activity of P450 2D1 were low and were similar to those of quinine and quinidine, which are specific inhibitors of P450 2D forms. The Ki value of mepyramine was especially low, at 34 nM. Furthermore, the effects of mepyramine on human P450 2D6 were investigated. Among the ten forms of human P450 expressed in yeast, mepyramine bound specifically to P450 2D6 in a binding assay. In human hepatic microsomes, mepyramine inhibited the debrisoquine 4-hydroxylation activity catalyzed by P450 2D6. These results indicate that histamine H1 receptor antagonists such as mepyramine are potent inhibitors of P450 2D forms because of their high affinity for these enzymes.