A series of possible transition state analogues of the glucuronidation reaction catalyzed by UDP-glucuronosyltransferase were tested for their inhibitory effect on glucuronidation of various substrates in a rat liver microsomal fraction. In general 4-nitrophenol glucuronidation was more effectively inhibited than that of 1-naphthol, bilirubin or testosterone. 2-(1-Naphthyl)ethyl-UDP and 2,2,2-(triphenyl)ethyl-UDP were the most effective inhibitors. Their inhibitory effect was competitive towards both UDP-glucuronic acid and 4-nitrophenol. These compounds were much more effective inhibitors than UDP; therefore addition of a lipophilic group enhances the inhibitory potency of UDP. The various UDP derivatives showed differences in their abilities to inhibit the glucuronidation of the four acceptor substrates, supporting the concept that the different forms of UDP-glucuronosyl transferase have different active sites.