Soil microorganisms were examined for their ability to grow on porcine gastric mucin as a sole source of carbon and energy. Streptomyces sp. OH-11242 thus selected was found to produce endo-alpha-N-acetylgalactosaminidase (endo-GalNAc-ase S), together with several mucin-degrading glycosidases. Endo-GalNAc-ase S is a new enzyme capable of hydrolyzing the innermost GalNAc-O-Ser (Thr) linkage of the mucin molecule. Studies on the fermentation conditions necessary for its production revealed that the enzyme was induced by mucin but the induction was inhibited by glucose and other easily assimilable carbon sources, as well as by complex nitrogen sources. Addition of palmitate, lambda-carrageenan, or crude mucin to a mucin-based production medium enhanced enzyme production and mycelial growth. When the initial mucin concentration of the production medium was increased, the maximum titer of endo-GalNAc-ase S produced in the culture both also increased, while the cultivation time giving the peak enzyme titer was prolonged. As a result of the studies, increased and reproducible production of endo-GalNAc-ase S was achieved, reaching 42 units/ml in a 100-ml culture and 31 units/ml in an 800-ml culture with 2 and 1.5% purified mucin, respectively, as the major carbon source.