The molecular form of UDP-glucuronosyltransferase involved in the catalysis of 3'-azido-3'-deoxythymidine (AZT)-5'-O-glucuronide was characterized in human liver microsomes. The specific activity (1.3 nmol/min per mg protein) in transplantable liver was more than 2-times higher than in post-mortem fragments. Liver microsomes from patients suffering Crigler-Najjar syndrome, who are genetically deficient in bilirubin UDP-glucuronosyltransferase, could also glucuronidate AZT to a similar extent, thus indicating that this protein was not involved in that process. A genetically engineered V79 cell line stably expressing a cDNA which encodes a human isozyme active towards 1-naphthol was unable to glucuronidate AZT. Clinically used drugs, most of them being glucuronidated, were tested as potential inhibitors of the glucuronidation of AZT in human liver microsomes. The drugs chemically related to 2-phenylpropionic acid, naproxen and flurbiprofen, and the steroid compounds testosterone, estrone and ethynylestradiol strongly inhibited AZT glucuronidation. Codeine and morphine also decreased the reaction rate although to a lower extent. Except estrone which elicited a partial competitive inhibition, ethynylestradiol, flurbiprofen naproxen and testosterone could competitively inhibit AZT glucuronidation with an apparent Ki of 38, 50, 172 and 250 microM, respectively. The results suggest that these drugs were substrates of the same isozyme(s) involved in AZT glucuronidation. Probenecid was a weak inhibitor of the reaction (Ki 900 microM), only when non-disrupted microsomes were used. This drug may compete with the anion carrier system involved in the microsomal uptake of UDP-glucuronic acid.