The secretory lysosomes of cytolytic lymphocytes house the principal apoptotic molecules for eliminating virus-infected cells: a membranolytic agent, perforin, and the serine protease, granzyme B. Perforin allows granzyme B access to cytosolic and nuclear substrates that, when cleaved, result in the characteristic apoptotic phenotype. Key among these substrates is a family of cytoplasmic caspases that mediate cell suicide. We have examined the caspase dependence of several nuclear and cytoplasmic parameters of apoptosis induced by purified perforin and granzyme B. Cell membrane leakage in response to perforin and granzyme B was independent of caspase activation; however, nuclear events such as DNA fragmentation and nuclear condensation and disintegration were abolished by the broad-acting caspase inhibitor, z-VAD-fmk. Despite being spared from nuclear damage, z-VAD-fmk-treated cells exposed to both cytotoxins uniformly died when they were re-cultured, while cells exposed to perforin or granzyme alone survived and proliferated as readily as untreated cells. Pretreatment of cells with z-VAD-fmk also resulted in reduced granzyme B nuclear uptake following addition of perforin; however, its uptake into the cytoplasm in the absence of perforin was unaffected. We conclude that cell death in response to perforin and granzyme B does not require caspase activation and still proceeds efficiently through non-nuclear pathways when nuclear substrate cleavage is inhibited.