Since preclinical studies indicated that 3'-azido-3'-deoxythymidine (AZT, zidovudine, Retrovir, BW A509U), a potent anti-HIV agent, is not metabolized extensively in rats, rabbits, mice, guinea pigs, cats, or dogs, the extensive biotransformation of AZT observed in humans was not expected. On average, approximately 75% of an oral AZT dose is recovered in human urine as a single metabolite while only 14-18% of the dose is recovered unchanged. Ultraviolet, infrared, nuclear magnetic resonance, and mass spectra and enzymatic degradation characterized the isolated major metabolite as a 5'-O-glucuronide (3'-azido-3'-deoxy-5'-beta-D-glucopyranuronosylthymidine, GAZT), a very unique nucleoside metabolite. These observations suggest that UDP-glucuronosyltransferase (UDPGT), EC2.4.1.17, mediates the in vivo biotransformation of AZT to GAZT. Since glucuronidation is one of the major conjugation reactions involved in the metabolic conversion of xenobiotics to more polar, water-soluble metabolites, it is an important detoxification pathway in humans. Therefore, it is important to understand the enzymatic basis for the discrepancy between metabolism of AZT in laboratory mammals and humans. This is especially relevant in light of the use of laboratory mammals to predict the metabolism of novel pharmaceutical agents in humans. The study presented herein confirms that liver UDPGT does catalyze the glucuronidation of AZT and that the higher substrate efficiency of AZT with human enzyme compared to rodent enzyme may account for metabolic differences observed in vivo.