A novel T cell subset characterized by cell surface NK1.1+ TCRαβ+ expression was investigated for its TCRα usage, particularly that of invariant Vα14 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 Vα14+and Vα14– subpopulations. Almost all of NK+ αβ thymocytes express Vα14 mRNA; however, only<20% were positive, while >80% were negative or undetectable for Vα14 TCR expression on the cell surface in the thymus. Similarly,∼50% of NK+ αβ T cells in spleen and bone marrow are Vα14+; 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 Vα14Jα281 gene with a 1 base N-region in all tissues. Thus, invariant Vα14 TCR is uniquely expressed on NK T cells, and can be a marker to distinguish NK, NK T and T cells.