Inhibition of heat shock protein 90 (HSP90) results in the degradation of oncoproteins that drive malignant progression, making HSP90 a target of cancer therapy. BIIB021, a synthetic HSP90 inhibitor, was being developed for the oral treatment of breast cancer. The objectives of this study were to obtain both quantitative and qualitative metabolite profiles of [14C]BIIB021 after incubations in rat, dog and human liver microsomes and hepatocytes. BIIB021 is extensively metabolized in both liver microsomes and hepatocytes. In liver microsomes for all species, the major metabolic pathway involves the monooxygenation on the methoxy-dimethylpyridine moiety to form M7, accounting for 35-53% of total radioactivity. M7 formation was also observed as a major metabolic pathway in human and rat hepatocytes, accounting for 37-49% of total radioactivity. However, in dog hepatocytes, the major metabolic pathways were due to the glucuronidation of M7 to form M4 accounting for 45% of total radioactivity and O-demethylation of methoxy-dimethylpyridine moiety to form M2 accounting for 26% of total radioactivity. Oxidative N-dealkylation (M11 and M12) is found solely in the rat. They represented 7% and 11% of the radioactivity in rat LM and hepatocytes, respectively. Non-enzymatic oxidative dechlorination of BIIB021 (M6) and its metabolites (M13 and M15) observed during the incubation was indicative of instability of BIIB021 chlorine moiety in aqueous solution. M6 and its derivative M15 were found dominant in human hepatocytes, representing 4% and 3.7% of the radioactivity, respectively. Direct displacement of chlorine on the amino-chloropurine moiety by glutathione to form M18 accounted for 9-13% of the radioactivity in rat and dog hepatocytes. Subsequent minor secondary metabolites M14 and M17 were observed in human, dog, and rat hepatocytes accounted for less than 10% radioactivity in each species. Mono-hydroxylation on the purine moiety (M10) was catalyzed probably via cytosolic enzyme aldehyde oxidase.