CD4+8- or CD4-8+ thymocytes have been regarded as direct progenitors of peripheral T cells. However, recently, we have found a novel NK1.1+ subpopulation with skewed T cell antigen receptor (TcR) V beta family among heat-stable antigen negative (HSA-) CD4+8- thymocytes. In the present study, we show that these NK1.1+ CD4+8- thymocytes, which represent a different lineage from the major NK1.1- CD4+8- thymocytes or CD4+ lymph node T cells, vigorously secrete interleukin (IL)-4 and interferon (IFN)-gamma upon stimulation with immobilized anti-TcR-alpha beta antibody. On the other hand, neither NK1.1- CD4+8- thymocytes nor CD4+ lymph node T cells produced substantial amounts of these lymphokines. A similar pattern of lymphokine secretion was observed with the NK1.1+ CD4+T cells obtained from bone marrow. The present findings elucidate the recent observations that HSA- CD4+8- thymocytes secrete a variety of lymphokines including IFN-gamma, IL-4, IL-5 and IL-10 before the CD4+8- thymocytes are exported from thymus. Our evidence indicates that NK1.1+ CD4+8- thymocytes are totally responsible for the specific lymphokine secretions observed in the HSA- CD4+8- thymocytes.