Recent studies have revealed that 10-20% of CD4+8- or CD4-8- thymocyte populations contain NK1.1+ T cell receptor (TCR)-alpha/beta+ cells. This subpopulation shows characteristics that are different from NK1.1- CD4+ or NK1.1- CD8+ T cells and seems to have developed in a manner different from NK1.1- T cells. Although extensive studies have been performed on the NK1.1+ TCR-alpha/beta+ thymocytes, the physiological role of the NK1.1+ TCR-alpha/beta+ thymocytes has been totally unclear. In the present study, we found that freshly isolated NK1.1+ TCR-alpha/beta+ thymocytes, but neither whole thymocytes nor lymph node T cells, directly killed CD4+8+ thymocytes from normal syngeneic or allogeneic mice by using a long-term cytotoxic assay in which flow cytometry was used to detect the cytotoxicity. However, only weak cytotoxicity was detected against thymocytes from lpr mice on which the Fas antigen that transduces signals for apoptosis into the cells is not expressed. Furthermore, the NK1.1+ TCR-alpha/beta+ thymocytes exhibited high cytotoxicity against T lymphoma targets transfected with fas genes as compared with the parental T lymphoma targets or target cells transfected with mutated fas genes, which lack the function of transducing signals. On the other hand, NK1.1+ effector thymocytes from gld mice that carry a point mutation in Fas ligand did not kill thymocyte targets from normal mice. The present findings, thus, consistently suggest that the NK1.1+ TCR-alpha/beta+ thymocytes kill a subpopulation among CD4+8+ thymocytes via Fas antigen and in this way regulate generation of T lineage cells in the thymus.