Regioselective 7-demethylation of scoparone is regularly employed as an indicator of phenobarbital-like induction of rat liver cytochrome P450 isoform CYP2B1, e,g,, by the antiepileptic drug phenytoin, After induction with phenobarbital and phenytoin, a new reaction sequence catalyzed by Cyp2c29 was identified in mouse liver microsomes. Cyp2c29-dependent 6-demethylation of scoparone resulted in the formation of isoscopoletin, an intermediate which is susceptible to further oxidation, This subsequent oxidation was also catalyzed by Cyp2c29 with a K-m of 30,31 muM and a V-max of 3,41 muM/min . muM P450, and resulted in the formation of the new metabolite 3-[4-methoxy-p-(3,6)-benzoquinone]-2-propenoate. This novel metabolite is the product of two consecutive oxidation reactions, proceeding over isoscopoletin to a putative lactone which is accessible to immediate hydrolysis, due to the onium character of the ring oxygen. This opening of the lactone ring corresponds to an oxidative hydrolysis. Differential oxidation of scoparone can be used as a sensitive indicator for distinguishing between different cytochrome P450 isoforms,