1. The metabolism of clonazepam was studied in vitro and in vivo using germ-free and ex-germ-free rats. 2. Incubation of clonazepam with rat-intestinal lumen contents gave nearly complete reduction of clonazepam to 7-aminoclonazepam. Rat-hepatic microsomes also reduced clonazepam but only under anaerobic conditions. Aerobic microsomal incubations gave 3-hydroxyclonazepam as the predominant metabolite. Both aerobic and anaerobic microsomal metabolism were induced by phenobarbital. Carbamazepine pretreatment significantly induced only 3-hydroxylation slightly; whereas naphthoflavone had no significant effect. 3. Extensive biliary disposition of hydroxylated clonazepam metabolites into the gut occurred. Only very low levels of clonazepam were found in bile. Using a linked-rat procedure enterohepatic recirculation of biliary metabolites was demonstrated and suppression (antibiotic treatment) or absence (germ-free) of the gut microflora nearly eliminated recycling. 4. Following oral administration of [14C]clonazepam to germ-free rats, reduced metabolites accounted for 15% of the radioactivity in the urine, with over 70% of the 14C attributed to a phenolic clonazepam metabolite. In contrast 77% of the recovered metabolites were derived from nitroreduction in the same animals following acquisition of an intestinal microflora; 7-acetamidoclonazepam was the major metabolite in these ex-germ-free animals. These studues show that clonazepam metabolism is primarily reductive in the presence of gut flora and oxidative in its absence.