Cystine depleted cystinotic fibroblasts incubated in cystine-free medium accumulate lysosomal-free cystine from the degradation of cystine-containing intracellular and extracellular proteins. In this report we have used this characteristic of these cells to study lysosomal proteolysis. We find that inhibitors of protein synthesis (cycloheximide, emetine, and puromycin) inhibit cystine accumulation from endogenous proteins and therefore act to inhibit lysosomal proteolysis of these proteins. However, cycloheximide does not inhibit cystine accumulation derived from the degradation of the extracellular disulfide-rich proteins, albumin and RNase, but lysosomal cystine accumulation derived from insulin is inhibited by cycloheximide. We conclude that a rapidly turning over protein may be required for the lysosomal degradation of intracellular and some extracellular proteins.