We have characterized the function, phenotype, ontogenic development, and T cell receptor (TCR) repertoire of a subpopulation of gamma delta thymocytes, initially defined by expressing low levels of Thy-1, that represents around 5% and 30% of total gamma delta thymocytes in adult C57BL/6 and DBA/2 mice, respectively. Activation of FACS-sorted Thy-1dull gamma delta thymocytes from DBA/2 mice with anti-gamma delta monoclonal antibodies in the presence of interleukin-2 (IL-2) results in the secretion of high levels of several cytokines, including interferon-gamma (IFN-gamma), IL-4, IL-10, and IL-3. In contrast, only IFN-gamma was detected in parallel cultures of Thy-1bright gamma delta thymocytes. Virtually all Thy-1dull gamma delta thymocytes express high levels of CD44 and low levels of the heat-stable antigen and CD62 ligand, while around half of them express the NK1.1 marker. Thy-1dull gamma delta thymocytes are barely detectable in newborn animals, and their representation increases considerably during the first 2 weeks of postnatal life. The majority of Thy-1dull gamma delta thymocytes from DBA/2 mice express TCR encoded by the V gamma 1 gene and a novel V delta 6 gene named V delta 6.4. Sequence analysis of these functionally rearranged gamma and delta genes revealed highly restricted V delta-D delta-J delta junctions, and somewhat more diverse V gamma-J gamma junctions. We conclude that Thy-1dull gamma delta thymocytes exhibit properties that are equivalent to those of natural killer TCR alpha beta T cells. Both cell populations produce the same distinct pattern of cytokines upon activation, share a number of phenotypic markers originally defined for activated or memory T cells, display similar postnatal kinetics of appearance in the thymus and express a very restricted TCR repertoire.