NK1+ double negative (DN) alphabeta T cells were present in the peritoneal exudate cells (PEC) of both normal and athymic B6 mice, accounting for as much as 25% of the total T cells, while their numbers were far less in the PEC of BALB/c and (BALB/c x B6)F1 mice. IL-2-dependent clones established from the DN alphabeta T cell population in the PEC of IL-2 receptor alpha-chain transgenic B6 mice exhibited potent cytotoxicity against a series of B cell lineage leukemias and myelomas, such as CD5+BCL1 and MOPC, without affecting NK-susceptible targets. The cytotoxicity of the clones against BCL1 and MOPC was specifically inhibited by anti-CD3, anti-alphabetaTCR, or anti-relevant Vbeta (Vbeta8) Ab, but not by control Abs, indicating that it was mediated by the specific alphabetaTCR/CD3. Other BALB/c-derived target cells expressing both MHC class I and class II were not affected, and neither Ab against them affected the cytotoxicity, strongly suggesting that the cytotoxicity of NK1+ DN alphabeta T cell clones was independent of the particular MHC Ags. The clones produced IFN-gamma, but little IL-2 or IL-4, in response to anti-CD3 stimulation, to the susceptible, but not resistant, targets, and to IL-12. The clones exhibited TCRalpha (Valpha8) distinct from an invariant TCRalpha (Valpha14) reported to dominate in thymic NK1+ alphabeta T cells. The presence of DN alphabeta T cells with similar functional features in the normal PEC was confirmed by the short term stimulation in vitro. The present results along with other recent reports strongly suggested that, like the mainstream alphabeta T cells, the NK1+ DN alphabeta T cell population consisted of functionally heterogeneous subsets.