An enzyme specific for oligosaccharides with type 1 chain was purified 7,000-fold from the culture broth of Streptomyces sp. 142. The enzyme, lacto-N-biosidase, was induced and secreted into culture medium when the strain was cultured in the presence of porcine stomach mucin. The enzyme was purified by anion-exchange chromatography on Q Sepharose, cation-exchange chromatography on S Sepharose, fast protein liquid chromatography on a Mono S column, and gel filtration chromatography on TSK gel HW55S. To remove contaminating alpha-1,3/4-fucosidase and beta-N-acetylglucosaminidase, final purification was done by fast protein liquid chromatography on a Mono S column and affinity chromatography on N-acetylglucosamine agarose. The purified enzyme gave only one major protein band with an apparent M(r) of 60,000 on sodium dodesyl sulfate-polyacrylamide gel electrophoresis. The enzyme had maximum activity at pH 5.5 and was stable at the pH range of 4.0-10.0. Substrate specificity studies with oligosaccharides labeled with 2-aminopyridine showed that the enzyme specifically hydrolyzed lacto-N-tetraose and the N-acetyllactosamine type of triantennary sugar chain with the type 1 chain, but did not hydrolyze type 2 chain oligosaccharides or the type 1 chain oligosaccharides with fucose or sialic acid including lacto-N-fucopentaose I and II and alpha-2,3-sialyl lacto-N-tetraose. The enzyme released lacto-N-biose from asialofetuin, and almost all oligosaccharides in asialofetuin were found to have only type 2 chains. Sequential digestion of extended type 1 chain oligosaccharides with alpha-1,3/4-fucosidase and lacto-N-biosidase was possible.