Interspecific somatic cell hybrids were analyzed by genetic complementation to determine if a lysosomal storage disease in sheep associated with deficiencies of beta-galactosidase and alpha-neuraminidase was homologous with any of four beta-galactosidase-deficient human diseases. Fibroblasts from beta-galactosidase-deficient sheep, cats, and human patients were fused and assayed histochemically for beta-galactosidase, with 5-bromo-4-chloro-3-indolyl beta-D-galactoside. We observed complementation in heterokaryons consisting of fibroblasts from beta-galactosidase-deficient sheep and fibroblasts from patients with galactosialidosis or mucolipidosis type II, but no complementation in heterokaryons consisting of fibroblasts from beta-galactosidase-deficient sheep and fibroblasts from human or feline GM1 gangliosidosis (type I) or from human mucopolysaccharidosis type IVB fibroblasts. We conclude that the ovine disease is due to a mutation at the genetic locus homologous with that of GM1 gangliosidosis and mucopolysaccharidosis type IVB, suggesting that the primary defect in the ovine disease is a mutation of the beta-galactosidase structural gene.