Coumarin is 7-hydroxylated by the P450 isoform Cyp2a-5 in mice and CYP2A6 in humans. Various drugs, endogenous substances, plant substances and carcinogens, altogether about 90 chemicals, were evaluated as possible inhibitors of coumarin 7-hydroxylase (COH) activity in mouse microsomes. The effects of selected compounds on COH activity in human liver microsomes were also tested. The furanocoumarin derivatives methoxsalen (8-methoxypsoralen) and psoralen proved to be the most potent inhibitors of mouse COH activity (IC50 values 1.0 and 3.1 microM, respectively). The furanocoumarins bergapten (5-methoxypsoralen), isopimpinellin (5,8-dimethoxypsoralen), imperatorin and sphondin also effectively inhibited mouse COH activity (IC50 values 19-40 microM). Methoxsalen, isopimpinellin and metyrapone were also inhibitors in mice in vivo. Methoxsalen was a potent inhibitor of COH activity also in human liver microsomes, (IC50 value 5.4 microM), whereas bergapten, isopimpinellin and imperatorin had no effect. The imidazole antimycotic miconazole was a potent but non-specific inhibitor of COH activity. Several known substrates and inhibitors of members in the CYP1A, CYP2B, CYP2C, CYP2D and CYP3A subfamilies were poor inhibitors of COH activity. These results suggest that (i) the coumarin-type compounds in particular interact with the active sites of Cyp2a-5 and CYP2A6, and (ii) the active sites of Cyp2a-5 and CYP2A6 are structurally different, since a number of compounds inhibited mouse, but not human COH activity.