The nature of the molecular defect resulting in the beta-galactosidase deficiency in different forms of GM1-gangliosidosis and mucopolysaccharidosis IV B (Morquio B syndrome) was investigated. Normal and mutant cultured skin fibroblasts were labeled in vivo with [3H]leucine and immunoprecipitation studies with human anti-beta-galactosidase antiserum were performed, followed by polyacrylamide gel electrophoresis and fluorography. In Morquio B syndrome, the mutation does not interfere with the normal processing and intralysosomal aggregation of beta-galactosidase. In cells from infantile and adult GM1-gangliosidosis, 85-kDa precursor beta-galactosidase was found to be synthesized normally but more than 90% of the enzyme was subsequently degraded at one of the early steps in posttranslational processing. The residual 5-10% beta-galactosidase activity in adult GM1-gangliosidosis is 64-kDa mature lysosomal enzyme with normal catalytic properties but with a reduced ability of the monomeric form to aggregate into high molecular weight multimers. Knowledge of the exact nature of the molecular defect underlying beta-galactosidase deficiency in man may lead to a better understanding of the clinical and pathological heterogeneity among patients with different types of GM1-gangliosidosis and Morquio B syndrome.