Expression of T-cell receptors of gamma/delta 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 gamma/delta MAbs it is possible to identify two distinct subsets of TCR gamma/delta+ cells that are characterized by a C gamma 1 or C gamma 2-encoded forms of gamma-chain, respectively. While the BB3 MAb-reactive (C gamma 1 encoded) cell subset is prevalent in peripheral blood (PB), these cells represent less than 10% in TCR gamma/delta+ thymocyte populations. In thymus, the majority of cells was found to react with delta-TCSI (or A13) MAbs. Culture of CD4-8- thymocytes (highly enriched in TCR gamma/delta+ cells) in IL-2 resulted in the de novo expression of CD8 surface antigen and of non MHC-restricted cytolytic activity. Cloning of CD4-8- thymocytes resulted, for the most part, in CD3+ TCR gamma/delta+ cells. Moreover, the majority of clones expressed the unusual delta-TCSI+ 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 P815 target cells and appropriate "stimulatory" antibodies was further employed for functional analysis of thymus-derived TCR gamma/delta+ clones. While anti-CD3 MAbs efficiently triggered the cytolytic activity of all clones irrespective of their phenotype, MAbs directed to TCR gamma/delta induced efficient lysis only of BB3+ or delta-TCSI+CD8 clones, but not of delta-TCSI+ CD8+ clones.(ABSTRACT TRUNCATED AT 250 WORDS)