The 10D1 Ag is a 90-kDa homodimeric molecule specifically expressed on a subpopulation of human T cells, and is involved in an alternative pathway of T cell activation. In the present study, we have examined the expression and function of the 10D1 Ag on human thymocytes. Three-color FMF analysis showed that the 10D1 Ag was highly expressed on minor but distinct subpopulations of double-negative and CD4 single-positive thymocytes, and weakly on a part of double-positive thymocytes, but not on CD8 single-positive thymocytes. In double-negative thymocytes, the vast majority of 10D1+ cells were immature thymocytes of CD7+2+3- phenotype. Interestingly, 10D1 mAb could induce the proliferation of CD4 single-positive thymocytes in the presence of goat anti-mouse Ig to cross-link the 10D1 Ag. The treatment of thymocytes with OKT4 mAb plus C but not with OKT8 mAb plus C totally abrogated the proliferative response induced by 10D1 mAb, indicating that the 10D1-responsible thymocytes were of CD4+8- phenotype. This 10D1 mAb-induced thymocyte proliferation was perfectly dependent on the endogenous IL-2/IL-2R system since a complete inhibition was observed with anti-IL-2 and anti-IL-2R mAb. The proliferating CD4 single positive thymocytes predominantly expressed the IL-2R alpha (p55) but not a detectable level of the IL-2R beta (p75). These results indicate that, although the 10D1 Ag can be detected on the CD7+2+3-4-8- thymocytes, its functional expression is restricted to a minor more mature CD4+ thymocyte population as well as in peripheral blood T cells, and the implications of these findings are discussed.