Using a combination of differential centrifugation and free flow electrophoresis (Harms, E., Kern, H., and Schneider, J. A. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 6139-6143) a single population of highly purified lysosomes was obtained from normal, I-cell disease type 1, and I-cell disease type 2 cultured fibroblasts. Our findings indicate that most of the residual acid hydrolase activities remaining within the I-cell fibroblasts are localized in the lysosomes, analogous to normal cells. Characterization of the carbohydrate-dependent properties of the lysosomal N-acetyl-beta-D-hexosaminidase revealed that the I-cell and normal enzymes do not contain a significant proportion of neuraminidase-susceptible sialic acid residues, interact poorly with the beta-galactose-specific lectin Ricinus communis and are highly sensitive to endohexosaminidase H treatment, indicating that the oligosaccharide units of both the I-cell and normal lysosomal N-acetyl-beta-D-hexosaminidase are predominantly of the high mannose type. The I-cell and normal lysosomal N-acetyl-beta-D-hexosaminidase, however, differed in their endocytotic properties. In contrast to the high rate of endocytosis of the normal lysosomal enzyme (7.8%/mg/h), the I-cell type 1 lysosomal enzyme failed to be endocytosed into Sandhoff cells indicating an absent or altered phosphohexyl recognition marker on the I-cell enzyme. Examination of the normal extracellular N-acetyl-beta-D-hexosaminidase revealed the presence of predominantly high mannose-type oligosaccharide units, similar to the corresponding lysosomal enzyme, although properties typical of complex-type oligosaccharide chains were also evident. In contrast, the secreted I-cell enzyme revealed the presence of oligosaccharide units predominantly of the complex type indicating that the I-cell N-acetyl-beta-D-hexosaminidase has had high mannose-type oligosaccharide chains modified to complex-type probably in the Golgi or GERL region prior to secretion from the cell.