A novel T cell subset characterized by cell surface NK1.1(+) TCR alpha beta(+) expression was investigated for its TCR alpha usage, particularly that of invariant V(alpha)14 TCR, which was found to be preferentially used in peripheral CD4(-)CD8(-) T cells developed at extrathymic sites. We found that NK+ alpha beta 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+ alpha beta T cells, two distinct subsets were detected; cell surface TCR V(alpha)14(+) and V(alpha)14(-) subpopulations, Almost all of NK+ alpha beta thymocytes express V(alpha)14 mRNA; however, only <20% were positive, while >80% were negative or undetectable for V(alpha)14 TCR expression on the cell surface in the thymus. Similarly, similar to 50% of NK+ alpha beta T cells in spleen and bone marrow are V(alpha)14(+) as revealed by FACS, TCR repertoire analysis by nucleotide sequences on inverse PCR products demonstrated that most NK+ alpha beta T cells express an invariant TCR encoded by the V(alpha)14J(alpha)281 gene with a 1 base N-region in all tissues. Thus, invariant V(alpha)14 TCR is uniquely expressed on NK T cells, and can be a marker to distinguish NK, NK T and T cells.