In the present report we describe a CD4+8- heat stable antigen-negative (HSA-) thymocyte subpopulation that expresses a distinguishably low density of alpha-beta-T-cell antigen receptors (TCR(lo)) from the majority of CD4+8- high-density TCR (TCR(hi)) mature-type thymocytes. This subpopulation appears relatively late in life. Analysis of MEL-14, Pgp-1 (CD44), ICAM-1 (CD54), and NK1.1 expression on this subpopulation revealed that the CD4+8- TCR(lo) population was a population having unique characteristics (MEL-14-, CD44+, ICAM-1+, and NK1.1+) compared to the CD4+8- TCR(hi) thymocytes, most of which are MEL-14+, CD44-, ICAM-1-, and NK1.1-. When TCR beta-chain variable region (Vp) usage was analyzed, this thymic population expressed predominantly products of V(beta-7) and V(beta-8.2) TCR gene families. Interestingly, cells with V(beta-8.1) TCRs, which are reactive to Mls-1a antigens, were not eliminated from the CD4+8- HSA- TCR(lo) subpopulation but had been eliminated from the major CD4+8- HSA- TCR(hi) subpopulation in Mls-1a strains. A subset with a phenotype similar to the CD4+8- HSA- TCR(lo) thymocytes was also identified primarily in bone marrow, and this subset constituted approximately half of the CD4+ T cells in the bone marrow. The CD4+8- HSA- TCR(lo) cells showed extremely high proliferative responses to immobilized anti-TCR antibody but generated negligible responses to allogeneic H-2 antigens compared to the responses generated by the major CD4+8- HSA- CD3(hi) cells. However, the CD4+8- HSA- TCR(lo) cells in Mls-1b mice mounted vigorous proliferative responses to Mls-1a antigens but not in Mls-1a mice. The properties of this T-cell subset suggest that these cells belong to a lineage distinct from the major T-cell population.