Self‐reactive clones, estimated by anti‐Vβ monoclonal antibodies (mAb) in conjunction with the Mls system, are confined to a population of intermediate (int) T‐cell receptor (TCR) (or CD3) cells (i.e. TCR^int cells), but are not found among TCR^high cells. The next questions to be answered are whether autologous killing is confined to TCR^int cells and how such killing is mediated. In this study, ⁵¹Cr‐labelled thymocytes of syngeneic or allogeneic origin were used as target cells (4‐hr assay). When liver and splenic mononuclear cells (MNC) obtained from B6 mice were used as effector cells, prominent autologous killing was seen in liver MNC, but not splenic MNC. Such killing was not seen when thymocytes from B6‐lpr/lpr mice (i.e. Fas⁻) were used as target cells, nor when liver MNC from MRL‐gld/gld mice (i.e. Fas ligand⁻) were used as effector cells (target thymocytes of MRL‐+/+ mice). Cell separation experiments using a cell sorter revealed that autologous killing was mediated for the most part by CD3^int cells, while allogeneic killing was mediated entirely by natural killer (NK) cells, TCR^int cells and TCR^high cells. Among CD3^int cells, the NK1.1⁺ subset (i.e. NK1.1⁺ T cells) manifested a higher level of autologous killing than did the NK1.1⁻ subset. Consistent with the results of a functional assay, it was found by reverse‐transcription–polymerase chain reaction (RT‐PCR) assay that CD3^int cells among liver MNC showed the expression of Fas ligand mRNA, while thymocytes expressed Fas mRNA. When class I major histocompatibility complex (MHC)⁻ thymocytes (from β₂‐microglobulin‐deficient mice) were used as target cells, NK cells, but not CD3^int cells, showed potent cytotoxicity. These results suggest that autologous killing is a major function of TCR^int cells with self‐reactivity, and that such killing is mediated by means of Fas ligand/Fas molecules.