Intra-gastric administration of brotizolam (0.1-200 mg/kg) daily for three days to rats resulted in no significant changes in the hepatic and intestinal cytochrome P-450-dependent or P-448-dependent mixed-function oxidases, or in the hepatic flavoprotein dimethylaniline N-oxidase. Liver microsomes from mouse, rat and man metabolized brotizolam by hydroxylation of the diazepine ring and of the methyl group at rates which were greater for mouse greater than rat greater than man. Brotizolam and its metabolites generated by rat-liver microsomes in vitro were not mutagenic in the Ames' test. Brotizolam, at 200 mg/kg per day for two to six weeks, depleted liver glutathione concentration and markedly increased liver gamma-glutamyl transpeptidase, glutathione reductase and glutathione transferase activities. Similar changes were not seen at the lower dose of 0.3 mg/kg. The observed increases in glutathione metabolism and the decreased tissue concentration of glutathione are indicative of high levels of glutathione conjugation, and provide a possible explanation for the equivocal increase in tumorigenicity seen in rats receiving brotizolam at high dosage.