We have studied the differentiation and repertoire selection during the maturation of CD4+CD8+ (DP) thymocytes into CD4+CD8- (CD4SP) and CD8+CD4- (CD8SP) T cells, in normal mice, mice transgenic for T cell receptor (TcR)-alpha beta restricted by either class I or class II major histocompatibility (MHC), and in mice deficient in class I or class II MHC expression. Our data suggest that mature CD4 and CD8 T cells derive from different pathways of T cell differentiation in the thymus. Thus, interaction of DP thymocytes with MHC class II leads to the immediate down-regulation of CD8, which occurs simultaneously with an increase in TcR expression; DPTcR(lo)HSA(hi) thymocytes mature into a CD4+CD8(lo) TcR(hi)HSA(hi) intermediate population. This cell population generates CD4SP thymocytes, the majority of which are still HSA(hi). In contrast, interaction with MHC class I induces the up-regulation of TcR, which precedes the down-regulation of CD4; DPTcR(lo) generate DPTcR(hi) thymocytes, the majority of which are the committed precursors of CD8SP cells. Further differentiation results in CD4 down-regulation and the transition from DPTcR(hi) into CD8+CD4(lo) TcR(hi)HSA(lo) and +D8SPTcR(hi)HSA- T cells. Since down-regulation of CD4 and CD8 occurs at different stages of thymocyte differentiation, our results do not support a stochastic/selective model of lineage commitment in the thymus.