1. The antitumour agent bropirimine undergoes significant Phase II conjugation in vivo. Incubation of [14C]bropirimine with human liver microsomes resulted in the formation of a single product peak (M1) using high-performance liquid chromatography with radiochemical detection and was tentatively assigned as bropirimine glucuronide based on sensitivity to beta-glucuronidase and by obtaining the expected mass of 442/444 amu with liquid chromatography/mass spectrometry. Following metabolite isolation, the structure of M1 was established as bropirimine O-glucuronide by 1H-nuclear magnetic spectroscopy. 2. Studies aimed at identifying the human liver UDP-glucuronosyltransferase (UGT) enzyme(s) involved in the glucuronidation of bropirimine were carried out using recombinant human UGTs and it was determined that glucuronidation of bropirimine was catalysed by UGT1A1, UGT1A3 and UGT1A9. Bropirimine O-glucuronidation followed Michaelis-Menten kinetics and the Km and Vmax (mean +/- SD; n = 3) were 1217 +/- 205 microM and 667 +/- 188 pmol min(-1) mg(-1), respectively. 3. The activity of bropirimine O-glucuronidation by human liver microsomes was inhibited by bilirubin (40%) and with mefenamic acid (80%). Although buprenorphine extensively inhibited the activity of bropirimine O-glucuronidation by UGT1A3, the inhibition profile did not parallel that observed in HLMs. 4. The results demonstrate that UGT1A9 and to a lesser extent UGT1A1 are responsible for the majority of bropirimine O-glucuronidation in man.