Ursodeoxycholic acid and its endogenous metabolite tauroursodeoxycholic acid inhibited in vitro the microsomal bilirubin UDP-glucuronosyltransferase from rat liver. The magnitude of the inhibition correlated well with the loss of integrity of microsomal vesicles, suggesting that bile salts needed to reach the lumen to exert their inhibitory effects. The endogenous bile acids cholic acid, chenodeoxycholic acid and deoxycholic acid also exhibited inhibitory effects on bilirubin glucuronidation in digitonin-disrupted microsomes. Ursodeoxycholic acid inhibitory capacity was similar to that of chenodeoxycholic acid and deoxycholic acid but greater than that of cholic acid, the major endogenous bile salt. Kinetic studies, performed in detergent-activated preparations, showed that the inhibitions produced by ursodeoxycholic and tauroursodeoxycholic acids were competitive toward both bilirubin and UDP-glucuronic acid. The estimated Ki(app) for both substrates did not differ statistically between ursodeoxycholic and tauroursodeoxycholic acids. Both bile salts were weak inhibitors toward bilirubin but rather strong inhibitors toward UDP-glucuronic acid.