Expression of T-cell receptors of gammddelta type characterizes a small subset of peripheral T lymphocytes which is homogeneously composed of cytolytic cells and, in most instances, lack CD4 and CD8 differentiation antigens. By the use of anti-TCR gammddelta MAbs it is possible to identify two distinct subsets of TCR gammddelta+ cells that are characterized by a Cy l or Cy2-encoded forms of gamma-chain, respectively. While the 863 MAb-reactive (Cy l encoded) cell subset is prevalent in peripheral blood (PB), these cells represent < 10% in TCR gammddelta+ thymocyte populations. In thymus, the majority of cells was found t o react with delta-TCS I (or A13) MAbs. Culture of CD4-8-thymocytes (highly enriched in TCR gammddelta+ cells) in IL-2 resulted in the de novo expression of CD8 surface antigen and of non MHC-restricted cytolytic activity. Cloning of CD4-8thymocytes resulted, for the most part in CD3'TCR gammddelta+ cells. Moreover, the majority of clones expressed the unusual delta-TCS I +CD8+' phenotype and lysed the NK-sensitive K562 target cells. Analysis of the immunoprecipitated TCR molecules showed the existence of the (rare) heavy (55 kDa) form of gamma-chain. A redirected killing assay using murine P8I 5 target cells and appropriate "stimulatory" antibodies was further employed for functional analysis of thymus-derived TCR gammddelta+ clones. While anti-CD3 MAbs efficiently triggered the cytolytic activity of all clones irrespective of their phenotype, MAbs directed to TCR gammddelta induced efficient lysis only of BB3+ or delta-TCSI+CD8-clones, but not of delta-TCSI+CD8+ clones. Therefore, it appears that a receptor type which appears to be relatively inefficient in mediating activation signals is predominant in the thymus and rare in the periphery.