Most human T cells express the TCR alpha/beta and either CD4 or CD8 molecules (single positive, SP); however, small numbers lack CD4 and CD8. In inbred mice, alpha/beta CD4-CD8- (double negative, DN) T cells preferentially express certain beta variable region (V beta) families and may arise via unique developmental pathways. Increased percentages of alpha/beta DN T cells have been identified in some human and murine autoimmune and immunodeficiency diseases. However, their contribution to disease pathology or normal immunity is unknown. To study the cell surface phenotype and TCR diversity of human alpha/beta DN T cells, these cells were isolated from the peripheral blood of healthy adults. The proportion of alpha/beta DN T cells expressing molecules associated with activation (HLA-DR), previous exposure to antigen (CD45RO), and cytotoxic function (CD56, CD57, and CD11b) was increased relative to SP T cells. The TCR V beta repertoire of alpha/beta DN T cells was different from that of alpha/beta SP T cells, although most major gene families were present. For example, higher proportions of V beta 11, a minor gene family in peripheral blood leukocytes, were found in most alpha/beta DN T-cell samples. In contrast to mice, no dominant V beta family was used consistently in different human individuals. Within an individual alpha/beta DN T cells possessed an oligoclonal TCR beta repertoire with conservation of several distinct junctional amino acid motifs with one joined to three different V beta genes in two individuals, suggesting that these cells have undergone a selection process driven by a limited set of ligands. The possibility that they may represent, at least in part, originally SP T cells anergized by down-modulation of CD4 or CD8 must also be entertained. Overall, this study demonstrates that human peripheral blood alpha/beta DN T cells possess unique phenotypic and TCR beta repertoire characteristics when compared with the major alpha/beta SP T cell populations and thus may serve specialized immunologic functions and/or have an unusual origin.