Human thymocytes lacking both CD4 and CD8 differentiation antigens were prepared by treating total thymocyte suspensions with a mixture of anti-CD4 and anti-CD8 monoclonal antibodies and complement. The resulting populations contained less than 2% CD4+, CD8+ or WT31+ cells and variable percentages (less than 20%) of CD3+ cells. These cell populations were cultured in recombinant IL-2 in the presence of peripheral blood mononuclear cells as feeder cells. Cells underwent extensive proliferation accompanied by a progressive increase of CD3+ and CD8+ cells. On the other hand, appearance of neither WT31+, alpha/beta-positive T cell receptor (TCR), nor CD4+ cells could be observed in several independent experiments. Functional analyses revealed the appearance and the progressive increase of cytolytic activity against the natural killer (NK)-sensitive K562 cells as well as the NK-resistant fresh melanoma cells. Experiments of T cell cloning indicated that both the expression of CD8 and CD3 antigens and the appearance of cytolytic activity were consequent to cell maturation occurring at the level of CD4-CD8- non-cytolytic cell precursors. In these experiments, more than 30% of cells underwent clonal expansion and all the clonal progenies obtained displayed cytolytic activity and expressed the CD3+WT31- surface phenotype. The expression of CD8 was variable, whereas no CD4+ clones could be obtained. Cells expressing such surface phenotype are known to belong to the TCR gamma-positive T lymphocyte subset lacking the typical alpha/beta TCR and thus appear to be the only T cell type capable of in vitro proliferation and maturation under easily reproducible culture conditions.