The role of beta-glucuronidase catalyzed hydrolysis of glucuronides on the in vivo disposition kinetics of xenobiotics was studied in the rat. The metabolic disposition kinetics of diflunisal, a compound undergoing transformation to an acyl and phenyl glucuronide, were studied in rats under control conditions and following administration of D-glucaro-1,4-lactone, a potent and specific beta-glucuronidase inhibitor. D-glucaro-1,4-lactone treatment resulted in a significant decrease in beta-glucuronidase activity in plasma, urine, and hepatic microsomes. Total (i.e. urinary and biliary) recovery of the acyl glucuronide following i.v. injection of diflunisal (10 mg/kg) was significantly higher in D-glucaro-1,4-lactone treated rats (41 +/- 3%, n=6) compared to control rats (29 +/- 2%, n=6) whereas for diflunisal phenyl glucuronide this total recovery was very similar in both groups of rats (16.0 +/- 1.0% vs. 18.0 +/- 0.2%, n=6, respectively). The partial clearance of diflunisal associated with the formation of the acyl glucuronide was significantly higher in D-glucaro-1,4-lactone treated rats (0.413 +/- 0.024 ml/min/kg) compared to control animals (0.269 +/- 0.042 ml/min/kg). The partial clearance related to the formation of the phenyl glucuronide, on the contrary, was not significantly affected by D-glucaro-1,4-lactone treatment. These results shows that the in vivo glucuronidation of diflunisal to the acyl glucuronide, unlike diflunisal glucuronidation to the phenyl glucuronide, is subject to a futile conjugation-deconjugation cycle. Such futile cycling may have significant therapeutic and toxic implications.