The metabolism of the steroidal aromatase inhibitor atamestane was studied in the rat, the cynomolgus monkey and in the human. Metabolite patterns were recorded in plasma, urine and bile (rat only) before and after enzymatic cleavage of sulfate and glucuronide conjugates. Atamestane was rapidly and extensively metabolized by all three species. Major metabolites which were observed in the human, could be isolated from urine pools of treated monkeys by preparative high performance liquid chromatography and were identified by GC/MS and 1H-NMR analysis. The metabolite patterns observed in the animals and in the human were similar, although some species- and sex-related differences were observed. There seem to be two principal routes by which atamestane is metabolized: one route is characterized by the attack of 17 beta-hydroxysteroid dehydrogenase, the other route includes hydroxylation of the 1-methyl group with subsequent attack by 5 beta-reductase, followed by a hydroxylation at position C-6. Some of the metabolites which were identified still had some pharmacological activity, although less marked than the parent compound.