beta-D-Galactosidase from Aspergillus niger was purified by conventional techniques, including the repeated use of chromatography on hydroxylapatite. The final preparation represented a 112-fold purification, with a 22% yield. The specific activity of the purified enzyme was 72 mumol of D-galactose released/min/mg of protein, using p-nitrophenyl beta-D-galactopyranoside as the substrate. The substrate specificity of the enzyme was studied by using saccharides having structural linkages similar to those found in naturally occurring glycoconjugates. At substrate concentrations of 5mM, the beta-D-galactosidase efficiently hydrolyzed beta-Gal-1 leads to OC6H4NO2-p, beta-Gal-(1 leads to 3)-Gal, beta-Gal-(1 leads to 3)-beta-Gal-1 leads to OC6H4NO2-p, and beta-Gal-(1 leads to 3)-alpha-Gal-1 leads to OC6H4NO2-p, at rates of 63, 53, 65, and 29 mumol/min/mg of protein, respectively. Slower hydrolysis was observed for beta-Gal-(1 leads to 4)-beta-Glc, beta-Gal-(1 leads to 4)-beta-GlcNAc-1 leads to OC6H4NO2-p, and beta-Gal-(1 leads to 6)-beta-GlcNAc-1 leads to OC6H4NO2-p, with rates of 10, 13 and 9 mumol/min/mg of protein, respectively. Poorly hydrolyzed, at rates 1/300th of that of beta-Gal-1 leads to OC6H4NO2-p, were synthetic substrates having D-galactose attached beta-(1 leads to 3)- to either GalNAc or GlcNAc. The Km value for beta-D-galactosidase with beta-Gal-(1 leads to 4)-beta-GlNAc-1 leads to OC6H4NO2-p was approximately 20 times that with beta-Gal-1 leads to OC6H4NO2-p. The beta-D-galactosidase of A. niger has a molecular weight of 300,000, as demonstrated by gel-filtration chromatography. Sodium dodecyl sulfate-poly(acrylamide)-gel electrophoresis indicated a single subunit having a molecular weight of 130,000.