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, approximately 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.