A simple procedure has been devised to isolate beta-galactosidase from jack bean meal. The final preparation gives one major protein banc in disc gel electrophoresis. The substrate specificity of this enzyme toward some natural oligosaccharides, glycoproteins, and sphingoglycolipids has been examined in detail. Among three isomers of N-acetyllactosamine, Galbeta1leads to4GlcNAc; while Galbeta1leads to3GlcNAc was hydrolyzed very slowly. This property can be used to distinguish the galactose linkage in asialo-GM1 (Galbeta1leads to3GalNAcbeta1leads to4Galbeta1leads to4Glcleads toCer) and that in lacto-N-neotetraosylceramide (Galbeta1leads to4GlcNAcbeta1leads to 3Galbeta1leads to4Glcleads toCer). For hydrolyzing glycolipids, the effect of sodium taurodeoxycholate and sodium taurochenodeoxycholate on the rate of hydrolysis was carefully examined. This enzyme hydrolyzes lactosylceramide and asialo-GM1 faster than GM1. These results suggest that in addition to the type and linkage of the penultimate sugar unit, the sugar unit at the distal position of the saccharide chain also affects the hydrolysis rate. It also readily liberates 80% D-galactosyl units from asialo alpha1-acid glycoprotein. Escherichia coli beta-galactosidase on the other hand cannot hydrolyze asialo-alpha1-acid glycoprotein, lactosylceramide, GM1, asialo-GM1, and lacto-N-neotetraosylceramide. The molecular weight of this enzyme is about 75,000 and the isoelectric point is pH 8.0. With p-nitrophenyl beta-D-galactopyranoside as substrate, optimal activity occurs at pH 2.8 with glycine-HCl buffer and at pH 3.5 with citrate-phosphate buffer. With lactose as substrate, the pH optimum in these two buffers are 2.8 and 4.0, respectively. Km values for p-nitrophenyl beta-D-galactopyranoside, o-nitrophenyl beta-D-galactopyranoside and lactose are 0.51 mM, 0.63 mM, and 12.23 mM, respectively. Many inhibitors for this enzyme including inorganic ions, monosaccharides, and glycosides are investigated. In contrast to E. coli beta-galactosidase, jack bean beta-galactosidase is not inhibited by p-aminophenyl thio-beta-D-galactopyranoside.