Murine cytotoxic T lymphocytes contain, in addition to the cytotoxic pore-forming protein perforin, another cytolytic factor localized in both cytoplasm and granules. Like perforin, this CTL cytotoxin lyses a variety of tumor cells; unlike perforin, it is stable in the presence of calcium, requires several hours to induce maximal lytic activity, and is antigenically related to the previously described tumor necrosis factor (TNF) and lymphotoxin (LT). However, it differs from TNF and LT in a number of biochemical and functional properties. TNF- and LT-specific cDNA probes did not hybridize with any CTL-specific message, indicating that the CTL cytotoxin is distinct from those two factors. It has an apparent Mr of 50 and 70 kd under reducing and nonreducing conditions, respectively, is secreted by secretagogue-stimulated CTLs, and causes DNA fragmentation in several targets, a phenomenon previously attributed to target cell damage by CTLs. These results suggest that killing by lymphocytes may encompass multiple mechanisms and polypeptides.