Long-term culture of mouse spleen cells in IL-3-conditioned medium induced a stable mast cell line. This mast cell line (MCL) which could not be cloned expressed NC-1.1, a receptor on cells which mediate natural cytotoxicity (NC), and CD32/CD16 but no markers of T cells, B cells, macrophages, or NK cells. The MCL cells were large and granular, with abundant cytoplasm and >90% stained with Alcian blue, a mast cell specific stain. Probing total RNA with cDNA encoding a mast cell-specific proteinase mMCP-5 identified the ~1-kb mMCP-5 transcript which was confirmed at the protein level. MCL cells mediated very high NC against WEHI-164 which increased with time in culture and which was blocked by anti-NC-1.1 but not by anti-TNFα. When incubated with WEHI-164 tumor cells, MCL cells showed transient induction of TNF-α mRNA, but no detectable surface protein. Thus long-term culture of murine spleen cells in IL-3 induces mast cells which express the NC-1.1 receptor of cells which mediate NC, and utilize a cytotoxic effector mechanism which is not TNF-α.