Two exo‐β‐galactosidases are involved in the lysosomal degradation of glycosphingolipids: GM1‐β‐galactosidase (EC 3.2.1.23) and galactosylceramidase (EC 3.2.1.46). Analyses were performed with both enzymes, using lactosylceramides with varying acyl chain lengths as substrates that were inserted into unilamellar liposomes and naturally occurring sphingolipid activator proteins sap‐B and sap‐C, rather than detergents, to stimulate the reaction.While sap‐B was a better activator for the reaction catalyzed by GM1‐β‐galactosidase, sap‐C preferentially stimulated lactosylceramide hydrolysis by galactosylceramidase. The enzymic hydrolysis of liposome‐integrated lactosylceramides was significantly dependent on the structure of the lipophilic aglycon moiety of the lactosylceramide decreasing with increasing length of its fatty acyl chain (C2>C4>C6>C8>C10>C18). However, in the presence of detergents the degradation rates were independent of the acyl chain length. Hydrolysis of liposomal lactosylceramide was compared with sap‐B‐stimulated hydrolysis of liposomal ganglioside GM1 by GM1‐β‐galactosidase and sap‐C‐stimulated degradation of liposomal galactosylceramide by galactosylceramidase.Kinetic and dilution experiments indicated that sap‐B forms water‐soluble complexes with both lactosylceramide and GM1. These complexes were recognized by GM1‐β‐galactosidase as optimal substrates in the same mode, as postulated for the hydrolysis of sulfatides by arylsulfatase A [Fischer, G. and Jatzkewitz, H. (1977) Biochim. Biophys. Acta 481, 561–572]. GM1‐β‐galactosidase was more active on these complexes than on glycolipids (GM1 and lactosylceramides) still residing in liposomal membranes. On the other hand, dilution experiments indicated that degradation of galactosylceramide and lactosylceramide by galactosylceramidase proceeds almost exclusively on liposomal surfaces: both activators, sap‐C and sap‐B, stimulated the hydrolysis of lactosylceramide analogues with long acyl chains more than the hydrolysis of lactosylceramides with short acyl chains.