Granzyme (gzm) A and gzmB have been implicated in Fas-independent nucleolytic and cytolytic processes exerted by cytotoxic T (Tc) cells, but the underlying mechanism(s) remains unclear. In this study, we compare the potential of Tc and natural killer (NK) cells of mice deficient in both gzmA and B (gzmA×B−/−) with those from single knockout mice deficient in gzmA (−/−), gzmB (−/−), or perforin (−/−) to induce nuclear damage and lysis in target cells. With the exception of perforin−/−, all in vitro– and ex vivo–derived Tc and NK cell populations from the mutant strains induced 51Cr-release in target cells at levels and with kinetics similar to those of normal mice. This contrasts with their capacity to induce apoptotic nuclear damage in target cells. In gzmA×B−/− mice, Tc/NK-mediated target cell DNA fragmentation was not observed, even after extended incubation periods (10 h), but was normal in gzmA-deficient and only impaired in gzmB-deficient mice in short-term (2–4 h), but not long-term (4–10 h), nucleolytic assays. This suggests that gzmA and B are critical for Tc/NK granule– mediated nucleolysis, with gzmB being the main contributor, while target cell lysis is due solely to perforin and independent of both proteases.