We have characterized CD4-CD8- double negative (DN) thymocytes that express TCR-alpha beta and represent a minor thymocyte subpopulation expressing a markedly skewed TCR repertoire. We found that DN TCR-alpha beta + thymocytes resemble mature T cells in that they (a) are phenotypically CD2hiCD5hiQa2+HSA-, (b) appear late in ontogeny, and (c) are susceptible to cyclosporin A-induced maturation arrest. In addition, we found that DNA sequences 5' to the CD8 alpha gene were demethylated relative to their germline state, suggesting that DN TCR-alpha beta + thymocytes are derived from cells that had at one time expressed their CD8 alpha gene locus. Because DN TCR-alpha beta + thymocytes are known to express an unusual TCR repertoire with significant overexpression of V beta 8, we were interested in examining the possible role played by self-Ag in shaping their TCR repertoire. It has been suggested that DN TCR-alpha beta + thymocytes are derived from potentially self-reactive thymocytes that have escaped clonal deletion by down-regulating their surface expression of CD4 and/or CD8 determinants. However, apparently inconsistent with such an hypothesis, we found that the frequency of DN thymocytes expressing various anti-self TCR (V beta 6, V beta 8.1, V beta 11, V beta 17a) were not increased in strains expressing their putative self-Ag, but instead were either unaffected or significantly reduced in those strains. With regard to V beta 8 expression among DN TCR-alpha beta + thymocytes, V beta 8 overexpression in DN TCR-alpha beta + thymocytes appeared to be independent of, and superimposed on, the developmental appearance of the basic DN thymocyte repertoire. Even though V beta 8 overexpression appeared to be generated by a mechanism distinct from that generating the rest of the DN TCR-alpha beta + thymocyte repertoire, we found that super-Ag against which V beta 8 TCR react introduced into the neonatal differentiation environment also significantly reduced, rather than increased, the frequency of DN TCR-alpha beta + V beta 8+ thymocytes. Thus, the present study is consistent with DN TCR-alpha beta + thymocytes being mature cells derived from CD8+ precursors, and documents that their TCR repertoire can be influenced, at least negatively, by either self-Ag or Ag introduced into the neonatal differentiation environment. However, we found no evidence to support the hypothesis that DN TCR-alpha beta + thymocytes are enriched in cells expressing TCR reactive against self-Ag.