Recombinant human liver UDP-glucuronosyltransferase (UGT), UGT1*6, which catalyzes the glucuronidation of small phenols, previously expressed in a V79 cell line (1) was photolabeled with [beta-32P]5N3UDP-glucuronic acid ([beta-32P]5N3UDP-GlcUA). Two polypeptides with an approximate molecular weight of 54 kDa were extensively photolabeled in the recombinant cell line while the nontransfected cell line showed no photoincorporation in this area. The identity of the two polypeptides as UGTs, which correspond to two different glycosylation forms of the same enzyme, was confirmed by Western blot using a polyclonal monospecific antibody directed against the 120 amino acids of the N-terminal end of UGT1*6. Preincubation with UDP-glucuronic acid (UDP-GlcUA) inhibited the photoincorporation of the probe into the polypeptides indicating competition of both the photoprobe and the nucleotide-sugar for the same binding site. It was further shown that photoincorporation of [beta-32P]5N3UDP-GlcUA into the UDP-GlcUA-binding site was saturable. The lack of photoincorporation of a related photoprobe, [beta-32P]5N3UDP-glucose ([beta-32P]5N3UDP-Glc), into UGT1*6 demonstrated specificity of this enzyme for UDP-GlcUA. In enzymatic assays, unlabeled 5N3UDP-GlcUA was shown to be an effective cosubstrate of the glucuronidation of 4-nitrophenol catalyzed by UGT1*6. The studies were further extended by demonstrating that photolabeling of UGT1*6 was inhibited by several active site-directed inhibitors. Finally, photoaffinity labelling was used in the purification of the labeled UGT1*6 using preparative gel electrophoresis. In conclusion, we have demonstrated that photoaffinity labeling with [beta-32P]5N3UDP-GlcUA is an effective tool for the characterization of enzymes such as recombinant UGTs that use UDP-GlcUA.