A novel T cell subset characterized by cell surface NK1.1⁺ TCRαβ⁺ expression was investigated for its TCRα usage, particularly that of invariant V_α14 TCR, which was found to be preferentially used in peripheral CD4⁻CD8⁻T cells developed at extrathymic sites. We found that NK₊ αβ T cell subsets account for 0.4% in thymocytes, 5% in the splenic T cells and 40.5% in the bone marrow T cells. Among these NK⁺ αβ T cells, two distinct subsets were detected; cell surface TCR V_α14⁺and V_α14^– subpopulations. Almost all of NK⁺ αβ thymocytes express V_α14 mRNA; however, only<20% were positive, while >80% were negative or undetectable for V_α14 TCR expression on the cell surface in the thymus. Similarly,∼50% of NK⁺ αβ T cells in spleen and bone marrow are V_α14⁺; as revealed by FACS. TCR repertoire analysis by nucleotide sequences on inverse PCR products demonstrated that most NK⁺ αβ T cells express an invariant TCR encoded by the V_α14J_α281 gene with a 1 base N-region in all tissues. Thus, invariant V_α14 TCR is uniquely expressed on NK T cells, and can be a marker to distinguish NK, NK T and T cells.