Bisnorbiotin and biotin sulfoxide are the major catabolites of biotin for humans, swine, and rats. Increased urinary excretion of bisnorbiotin, biotin sulfoxide, or both have been observed during pregnancy and in patients treated with certain anticonvulsants. We sought more insight into the sites and mechanisms of biotin catabolism by exposing rats in vivo to compounds known to induce classes of enzymes that were candidates to catalyze the biotransformations. Rats were treated with the anticonvulsants phenytoin, phenobarbital, and carbamazepine, the steroid hormones dexamethasone and dehydroepiandrosterone, and the peroxisome proliferators clofibrate and di(2-ethylhexyl)phthalate. [14C]Biotin was injected intraperitoneally at physiologic doses in treated rats and control rats; HPLC and radiometric flow detection were used to specifically identify and quantify [14C]biotin and its metabolites in urine. Treatment effects were assessed by the change in the urinary excretion of [14C]bisnorbiotin and [14C]biotin sulfoxide in response to administration of [14C]biotin. No significant changes resulted from treatment with any of the anticonvulsants. With the steroid hormones and the peroxisome proliferators, [14C]bisnorbiotin excretion increased significantly. These results indicate that biotin is converted into bisnorbiotin in the liver and that this conversion likely occurs in peroxisomes or mitochondria or both via beta-oxidative cleavage, and, in contrast to responses in humans, the enzymes responsible for the formation of biotin sulfoxide in rats are not induced by the anticonvulsants examined here.