The oxidative removal of two different methyl groups from scoparone by a cytochrome P-450-mediated reaction proceeds with greater velocity for the 6-methyl group than for the 7-methyl group. In vivo experiments in mice were carried out with the two respective radioisomers [6-methyl-14C]scoparone and [7-methyl-14C]scoparone. The consecutive administration of the scoparone to the same animal is followed by collecting the 14CO2 exhaled (breath test). The maximal exhalation rate, measured 4 min after i.p. administration of the substrates, is four times greater for [6-methyl-14C]scoparone than for the [7-methyl-14C]scoparone. The total recovery of radioactivity in the whole animal (except skin) including the exhaled 14CO2 is 86% for [7-methyl-14C]scoparone and 84% for [6-methyl-14C]scoparone. Further analysis shows that the proportion of radioactivity found in various excretory routes is different; however, in the case of the poorly exhaled [7-methyl-14C]scoparone, almost twice as much radioactivity is found in bile and urine than for the more rapidly exhaled [6-methyl-14C]scoparone, whereas liver and other organs show little changes. Pretreatment of animals with various inducers, such as phenobarbital, 3-methylcholanthrene, warfarin and cobaltous chloride, characteristically affects the ratio of the two demethylations of scoparone. In the case of warfarin pretreatment, the ratio of the 6- to 7-demethylation is elevated to nearly 6, after cobaltous chloride the ratio is lowered to 2.8, whereas phenobarbital and 3-methylcholanthrene have no effect. These results may provide possibilities of noninvasive in vivo recognition of the inductive state of a pretreated animal.