UDP-glucuronosyltransferase 2B7 (UGT2B7) is involved in the glucuronidation of a wide array of clinically important drugs and endogenous compounds in humans. The aim of this study was to identify an isoform-selective probe substrate that could be used to investigate genetic and environmental influences on glucuronidation mediated by UGT2B7. Three potential probe substrates [3'-azido-3'-deoxythymidine (AZT), morphine, and codeine], were evaluated using recombinant UGTs and human liver microsomes (HLMs; n = 54). Of 11 different UGTs screened, UGT2B7 was the principal isoform mediating AZT glucuronidation, morphine-3-glucuronidation, and morphine-6-glucuronidation. Codeine was glucuronidated equally well by UGT2B4 and UGT2B7. Enzyme kinetic analysis of these activities typically showed higher apparent Km values for HLMs (pooled and individual) compared with UGT2B7. This difference was least (less than 2-fold higher Km) for AZT glucuronidation and greatest (3- to 6-fold higher Km) for codeine glucuronidation. Microsomal UGT2B7 protein content correlated well with AZT glucuronidation (rs = 0.77), to a lesser extent with morphine-3-glucuronidation (rs = 0.50) and morphine-6-glucuronidation (rs = 0.51), but very weakly with codeine glucuronidation (rs = 0.33). Livers were also genotyped for the UGT2B7*2 (H268Y) polymorphism. No effect of genotype on microsomal glucuronidation or UGT2B7 protein content was observed. In conclusion, although both AZT and morphine can serve as in vitro probe substrates for UGT2B7, AZT appears to be more selective than morphine. Codeine is not a useful UGT2B7 probe substrate because of significant glucuronidation by UGT2B4. The UGT2B7*2 polymorphism is not a determinant of glucuronidation of AZT, morphine, or codeine in HLMs.