The major beta-galactosidase of rabbit brain has been purified over 400-fold. The enzyme converts G-M-1-ganglioside; Gal beta-1 yields 3 GalNAc beta-1 yields 4 (NANalpha-2 yields 3) Gal beta-1 yields 4 Glc yields ceramide (G-M-1) into Tay Sachs ganglioside GalNAc beta-1 yields 4 (NANalpha-2 yields 3) Gal beta-1 yields 4 Glc yields ceramide (G-M-2-ganglioside) and ceramide lactoside, Gal beta-1 yields 4 Glc yields ceramide (Gal-Glc-Cer) into glucocerebroside, Glc yields ceramide (Glc-Cer). The enzyme also hydrolyzes the synthetic substrates NPh-Gal and MeUmb-Gal. It is eluted as a single peak from Sephadex G-200 columns when natural and synthetic substrates were used and has an isoelectric point of 6.3. We were unable to resolve activity towards G-M-1-ganglioside and Gal-Glc-Cer by polyacrylamide electrophoresis in two buffer systems. With G-M-1 the pH optimum was 4.3 in acetate buffer and the K-m value 78 mu-M while with Gal-Glc-Cer, a pH optimum of 4.5 and a K-m of 17 mu-M were found. Hydrolysis of both natural and synthetic substrates was inhibited by gamma-D-galactonolactone, D-galactose and lactose. The data strongly suggest that a single beta-galactosidase hydrolyzes all the substrates tested.