The metabolism of brotizolam (2-bromo-4-(2-chlorophenyl)-9-methyl-6H-thieno [3,2-f]-1,2,4-triazolo[4,3-a]-1,4-diazepine, We 941, Lendormin) was studied in the bile of the rat and in the urine of the dog, rhesus monkey and man using 14C-labeled substance. Concentration, fractionation and purification of the metabolites were performed using thin layer chromatography, column chromatography or high pressure liquid chromatography. Metabolites were structurally characterized by thin layer chromatography, high pressure liquid chromatography, mass spectrometry and nuclear magnetic resonance spectrometry using reference compounds. Hydroxylation at different sites of the brotizolam molecule and subsequent conjugation were the metabolic pathways preferred by far in the species studied. Unchanged brotizolam was excreted in minute amounts only, if at all. In man and monkey We 964 (brotizolam hydroxylated in the methyl group) and We 1061 (brotizolam hydroxylated in the diazepine ring) represented the main metabolites. In the rat, the main metabolites were We 1061 and a brotizolam hydroxylated in the phenyl ring. The main metabolites found in the dog were We 964 and We 1064, an isomeric compound of We 1061. Since We 1061 is irreversibly transformed in an alkaline medium to We 1064, the latter could be formed due to the clean-up processes. Thus, in the dog also We 1061 was probably the metabolite which was actually excreted renally. The proposed structures of minor metabolites are presented.