The beta-glucuronidase activities of bacterial strains isolated from the rat intestinal tract were studied both in vitro in culture media and in vivo in the intestinal contents of gnotobiotic rats. Only 50 of 407 strains tested were found to be positive in vitro. They belonged to the three genera Clostridium, Peptostreptococcus, and Staphylococcus. The in vitro-negative strains were also negative in vivo. The beta-glucuronidase activities of the beta-glucuronidase activities of the positive strains were generally greater in vivo than in vitro. The highest in vivo activities were found in the intact bacterial cells and in the soluble fractions prepared from disrupted pellets. There was a discrepancy between the activities obtained from both conventional and gnotobiotic rats harboring selected positive strains, suggesting that the main beta-glucuronidase-positive strains have not yet been isolated from the intestines of conventional rats.