βGalactosidase [EC 3.2.1.23] from jack bean meal was purified by heat treatment, followed by Sephadex G-200 and DEAE-Sephadex column chromatography. β-Galactosidase from almond emulsin was purified by CM-Sephadex and Sephadex G-200 column chromatography. β-Galactosidase from almond contained β-glucosidase [EC 3.2.1.21] activity,but β-galactosidase from jack bean did not. Both enzyme preparations specifically released galactose from IgG glycopeptide labeled with radioactive sugars. Aglycon specificities of the purified β-galactosidases were examined by using NaB3H4-reduced milk oligosaccharides. The purified β-galactosidases did not act on terminal β-galactosidic residues in lacto-N-fucopentaitol II and in lacto-N-fucopentaitol III. β-Galactosidase from jack bean meal hydrolyzed Galβ1→4GlcNAc linkage in lacto-N-neotetraitol about 50 times faster than Galβ1→3GlcNAc linkage in lacto-N-tetraitol at a substrate concentration of 14 μM. β-Galactosidase from almond hydrolyzed the Galβl→4GlcNAc linkage about 13 times faster than the Galβ1→3GlcNAc linkage under the same conditions. It was possible to cleave more than 90% of the Galβ1→4GlcNAc linkage without any significant hydrolysis of the Galβ1→3GlcNAc linkage using β-galactosidase from jack bean meal.