Lysosomes were isolated from Chinese hamster ovary cells by fractionation of a postnuclear supernatant in consecutive density gradients. By marker enzyme analysis, the preparation was 63-fold enriched for lysosomes compared to the homogenate and contained at most trace amounts of marker activities for plasma membrane, Golgi, endoplasmic reticulum, peroxisomes, cytosol, and mitochondria. The lysosomes were intact as indicated by greater than 95% latency of beta-hexosaminidase activity, and the yield was about 12% relative to the homogenate. By electron microscopy, the lysosomal preparation contained very few mitochondrial profiles. By cytochemistry, greater than 80% of the organelle profiles were positive for the native lysosomal marker, acid phosphatase, and profiles were positive for long-term internalized horseradish peroxidase, an endocytic marker for lysosomes. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the lysosomal preparation displayed a unique pattern of polypeptides and was devoid of mitochondrial contamination. Lysosomes were fractionated into membrane and lumenal compartments by Na2CO3 treatment. Each compartment contained 20-30 distinct electrophoretic species ranging from 18 to 200 kDa. Each polypeptide could be assigned to either the membrane or lumenal compartment. A comparison of silver-stained polypeptides with those metabolically labeled with [35S]methionine indicated that, with the possible exception of an 18-kDa species, all of the major lysosomal polypeptides in both compartments were derived by endogenous synthesis in these exponentially growing fibroblasts.