Objective— Lysosomal proteinases have been implicated in a number of pathologies associated with extracellular matrix breakdown. Therefore, we investigated the possibility that the lysosomal proteinase cathepsin S may be involved in atherosclerotic plaque destabilization. Methods and Results— Atherosclerotic plaques in the brachiocephalic arteries of fat-fed apolipoprotein E/cathepsin S double knockout mice had 73% fewer acute plaque ruptures ( P =0.026) and were 46% smaller ( P =0.025) than those in age-, strain-, and sex-matched apolipoprotein E single knockout controls. When the incidence of acute plaque rupture was normalized for plaque size, the reduction in the double knockouts was 72% ( P =0.039). The number of buried fibrous layers, indicative of an unstable plaque phenotype, was reduced by 67% in the double knockouts ( P =0.008). The cysteine proteinase inhibitor, egg white cystatin, was biotinylated and used as an active-site-directed probe for cathepsins. Biotinylated cystatin selectively detected cathepsin S in extracts of human carotid atherosclerotic plaque. Active cathepsin S was detectable in extracts of human atherosclerotic plaque but not in nondiseased carotid arteries. Active cathepsins were especially prominent in macrophages in the shoulder regions of plaques, areas considered to be vulnerable to rupture. Cathepsin S protein colocalized with regions of elastin degradation in human coronary plaques. Conclusion— These data provide direct evidence that an endogenous proteinase, cathepsin S, plays an important role in atherosclerotic plaque destabilization and rupture.