Signals transduced through the T cell antigen receptor (TCR) are modulated by the src family tyrosine kinase p56lck (lck), which associates in mature T cells with the coreceptor molecules CD4 and CD8. Here we describe a novel function of lck in immature CD4+CD8+ thymocytes, that of regulating TCR expression. Activation of lck in immature CD4+CD8+ thymocytes by intrathymic engagement of CD4 maintains low TCR expression by causing most TCR components to be retained and degraded within the endoplasmic reticulum. Importantly, activation of lck in immature CD4+CD8+ thymocytes results from engagement of surface CD4 molecules, but not surface CD8 molecules, despite the nearly fourfold greater surface expression of CD8 than CD4. The competence of CD4 to activate lck in CD4+CD8+ thymocytes relates to the fact that a relatively large fraction of surface CD4 molecules (25-50%) are associated with intracellular lck molecules, whereas only 2% of surface CD8 molecules are associated with lck. The amount of lck associated with CD4 in CD4+CD8+ thymocytes is diminished by chronic CD4 engagement in the thymus, as activated lck molecules subsequently dissociate from CD4. Indeed, the amount of lck associated with CD4 in CD4+CD8+ thymocytes is markedly increased in major histocompatibility complex (MHC) class II- mice that lack the intrathymic ligand for CD4 and in which surface CD4 molecules are consequently not engaged. Thus, the present study demonstrates that (a) activation of lck in CD4+CD8+ thymocytes regulates distribution and expression of TCR components; (b) unlike CD4 molecules, CD8 molecules on CD4+CD8+ thymocytes cannot efficiently activate lck despite their significantly greater surface expression; and (c) the amount of lck associated with CD4 in the CD4+CD8+ thymocytes is inversely related to the extent of CD4 engagement by MHC class II molecules in the thymus.