The role of mast cells and mast-cell-derived factors in natural cytotoxic reactions was investigated. Cultured and freshly isolated murine mast cells are shown to be cytotoxic to WEHI-164 and YAC-1 targets in 18-hr viability assays but not in 4-hr assays. Here, we describe a cytotoxic factor in murine mast cells that is immunologically related to tumor necrosis factor (TNF). This TNF-like factor lyses WEHI-164 cells with a slow time course requiring 16-20 hr for the lytic reaction to complete. Antibodies specific for human and murine TNF and human lymphotoxin partially block mast cell lysis of WEHI-164 cells. These antibodies react on immunoblots with one major mast cell protein band of 50 kDa. Immunoblot analysis shows this factor in cloned and uncloned cultured mouse mast cells and in mature "connective tissue-type" mast cells freshly purified from rat or mouse peritoneal cavities. The amount of this factor is greatly enhanced in cells that have been stimulated with a combination of phorbol ester/concanavalin A or bacterial lipopolysaccharide. Subcellular fractionation analysis of mast cells with Percoll gradients reveals two pools of TNF-related cytotoxic activity that are associated with free cytosolic material and granule fractions. In contrast to cytotoxic T lymphocytes and natural killer cells, granule-enriched fractions of mast cells do not contain any hemolytic activity. The localization of the TNF-like molecule in mast cell granules may play a strategical role in the rapid delivery of this mediator to the target cell membrane following cell surface stimulation and degranulation.