1. In the rat, scoparone (6,7-dimethoxycoumarin) is regioselectively O-demethylated in vitro by the hepatic cytochrome P450 (P450) system, yielding isoscopoletin and scopoletin. 2. Scoparone has been proposed as an indicator substrate for the assessment of P450 differentiation in vitro in rat. It has been suggested that isoscopoletin formation mainly reflects activity of enzymes of the P4501A subfamily, whereas scopoletin formation has been associated with P4502B activity. 3. In the present study, rate of formation of scopoletin and isoscopoletin were measured in hepatic microsomes from male, female and castrated male rats, in castrates treated with testosterone, in males treated with oestradiol, and in females treated with testosterone. Furthermore, effects of induction by phenobarbital (PB), beta-naphthoflavone (BNF), isoniazid, triacetyloleandomycin, and dexamethasone were studied in both sexes. 4. Scoparone metabolism was partly sex- and steroid-dependent. Variation of isoscopoletin formation with sex or hormonal status correlated well with ethylmorphine demethylation. 5. Scoparone-O-demethylation was regioselectively induced by PB and BNF. Induction effects were not very large and showed no sex differences. 6. Microsomal metabolism of scoparone was partly inhibited by a monoclonal antibody against P4502C11. Scopoletin and isoscopoletin formation were inhibited when ethylmorphine was added to the incubation mixture. Scoparone competitively inhibited testosterone 6 beta-hydroxylation. 7. It is concluded that a number of P450 enzymes, including those which are constitutively expressed, contribute to the biotransformation of scoparone. This lack of selectivity limits the usefulness of scoparone as a general indicator substrate for P450 differentiation in rats.