The purpose of our investigation was to determine whether iopanoyl glucuronide, the major metabolite of iopanoic acid (Telepaque), undergoes hydrolysis by bacterial beta-glucuronidase in dogs. The conjugated compound was identified and quantitated by elemental analysis, fluorescent excitation analysis, thin-layer chromatography, and high pressure liquid chromatography. The experiments were performed before and after combined antibiotic treatment with neomycin and vancomycin. It was first determined that reabsorption and excretion of sodium iopanoate was only minimally diminished during antibiotic treatment. Known amounts of iopanoyl glucuronide were infused into the small bowel of 4 awake dogs with chronic bile fistula, and bile was collected for 5--8 hours. The excretion of the recirculated conjugated compound was 4--5 times lower during antibiotic treatment. Incubation of ileal fluid with bile containing iopanoyl glucuronide suggested that beta-glucuronidase hydrolyzes the conjugated compound. Hydrolysis was markedly decreased after pretreatment with antibiotics. These findings suggest that the beta-glucuronidase produced by bacteria may be a major mechanism in enterohepatic recirculation of iopanoyl glucuronide. Mechanisms and possible implications are discussed.