Bovine brain phosphatidylserine effectively activates human brain galactosylceramidase (Hanada, E. and Suzuki, K. (1979) Biochim. Biophys. Acta 575, 410-420). Its effect on the other beta-galactosidase (Gm1-ganglioside beta-galactosidase) in human tissues, genetically distinct from galactosylceramidase, was examined. When partially purified human brain beta-galactosidase preparations, pure with respect to each other, were used as the enzyme source and when lactosylceramide, a common glycosphingolipid substrate for both beta-galactosidases, was used as the substrate, phosphatidylserine activated only hydrolysis of lactosylceramide by galactosylceramidase but not by GM1-ganglioside beta-galactosidase. With either galactosylceramide or lactosylceramide as substrate, and with phosphatidylserine as the activator, diagnosis of globoid cell leukodystrophy was possible using whole homogenates of cultured fibroblasts. Since 80-90% of lactosylceramide-cleaving activity in normal fibroblasts is due to GM1-ganglioside beta-galactosidase and since fibroblasts of globoid cell leukodystrophy patients are genetically deficient in galactosylceramidase but normal in GM1-ganglioside beta-galactosidase, these rsults are also consistent with specific activation of galactosylceramidase by phosphatidylserine.