Cathepsin Z is a cysteine proteinase found in lysosomes of human cells. It was detected in human cultured cell lines using the peptidyl diazomethane inhibitor Fmoc-Leu-Leu- [¹²⁵]Tyr-CHN₂. The labeling of cathepsin Z by the inhibitor was both time- and concentration-dependent, and the proteinase was found in all human cell lines examined. The characteristics of cathepsin Z were examined in U-937 cells, a human monocytic line. The labeling of cathepsin Z was blocked by pre-incubation of the cells either in non-iodinated inhibitor or in the epoxysuccinyl peptide inhibitor E-64d, a specific inhibitor of cysteine proteinases. Cathepsin Z was not immunoprecipitated by antisera specific for cathepsins B, L, or S. Cathepsin Z has been estimated to be at millimolar concentrations in lysosomes, suggesting that it is a major lysosomal proteinase. The molecular weight of cathepsin Z was calculated to be 22.4 kDa by SDS-PAGE and 45— 47 kDa by native PAGE and gel exclusion chromatography, indicating that it is dimeric. Cathepsin Z is susceptible to digestion by endoglycosidase H, and oligosaccharides comprise 3.1 kDa of the reduced molecular weight. The expression of cathepsin Z was not affected by differentiation of U-937 cells with phorbol ester, unlike the expression of cathepsins B and S. Undifferentiated U-937 cells express low levels of cathepsins B and S; after differentiation, expression of cathepsins B and S is greatly increased. Cathepsin Z was purified from U-937 cells by anion exchange chromatography (Mono Q), affinity chromatography (concanavalin A), and preparatory electrophoresis. N-terminal sequence analysis of both the purified protein and fragments of the protein from a V8 digest indicates that cathepsin Z is a member of the papain superfamily of cysteine proteinases.