R ecent work indicates that subpopulations of human fecal bacteria, averaging ∼1% of the total viable fecal flora, degrade the oligosaccharide side chains of hog gastric mucin, which structurally resembles human epithelial mucins. Here we report studies to determine whether degradation of mucin oligosaccharides is related to glycosidase production by bacteria growing in anaerobic fecal cultures. Triplicate cultures containing hog gastric mucin were inoculated with serially diluted feces from each of seven healthy subjects. When the stationary growth phase was attained, mucin oligosaccharide degradation and both cell-bound and extracellular activities of four glycosidases were measured in each culture. Cell-bound β-d-galactosidase, β-N-acetylglucosaminidase, and sialidase were present in bacteria growing at all levels of fecal inocula, including 10⁻¹¹ g. In contrast, extracellular activities were present in every culture inoculated with 10⁻⁴−10⁻⁷ g feces, but were diminished or absent in cultures inoculated with 10⁻⁸−10⁻¹¹ g feces. Bacterial autolysis was an unlikely cause of extracellular glycosidase activity, since p-nitrophenyl-α-l-fucosidase remained cell bound in cultures at every level of fecal inoculum. Degradation of mucin oligosaccharides was associated with extracellular, but not with cell-bound β-d-galactosidase, β-N-acetylglucosaminidase, and sialidase. Among the seven subjects, the estimated most probable numbers (MPN) of fecal bacteria producing extracellular β-d-galactosidase, β-N-acetylglucosaminidase, and sialidase ranged from 10⁶−10¹⁰/g dry fecal wt, were comparable to the MPN of mucin-degrading bacteria, and were significantly smaller than the MPN of total fecal bacteria.We interpret these findings as evidence for the existence of bacterial subpopulations in the normal fecal flora that produce extracellular glycosidases, and that these subpopulations have a major role in degrading the complex oligosaccharides of mucin in the gut lumen.