alpha-L-Fucosidase (alpha-L-fucoside fucohydrolase; EC 3.2.1.51) preparations from Streptomyces sp. 142 were found to contain an enzyme specific for lacto-N-biosidic (Gal beta 1-3GlcNAc beta 1-) linkages (type 1 structure) in oligosaccharides. The enzyme preparation, which was eluted after alpha-fucosidase from a CM-Sepharose column, contained some alpha-fucosidase activity but was free from other glycosidases and proteases. Substrate specificity studies with oligosaccharides labeled with 2-aminopyridine showed that the enzyme specifically hydrolyzed lacto-N-tetraose (Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc) but did not hydrolyze lacto-N-neotetraose (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc), lacto-N-triose, sialyl lacto-N-tetraose, lacto-N-fucopentaose I, II, or III, asialo-GM1 tetrasaccharide, or poly-N-acetyllactosamine. Structural analysis of the enzyme digest of the N-acetyllactosamine type of triantennary sugar chain with type 1 structure showed that lacto-N-biose (Gal beta 1-3GlcNAc) and the N-acetyllactosamine type of biantennary sugar chain were produced. Thus this enzyme was tentatively named lacto-N-biosidase, because it hydrolyzes oligosaccharides containing a type 1 structure at the nonreducing terminus and produces lacto-N-biose.