CD4 is a cell surface glycoprotein expressed by a subset of T lymphocytes and functions to enhance T-cell activation. CD4 is noncovalently associated via the cytoplasmic domain with the protein-tyrosine kinase p56(lck), a member of the src protein-tyrosine kinase family. Upon activation of protein kinase C by phorbol ester, CD4 is phosphorylated on cytoplasmic serine residues and internalized from the cell surface, and disruption of the CD4- p56(lck) complex occurs. The exact relationship between these events is likely to be functionally significant, as cytoplasmic-domain serine phosphorylation and internalization have been shown to regulate the function of receptors that possess intrinsic protein-tyrosine kinase activity. Here we demonstrate that p56(lck) slows the rate of phorbol 12-myristate 13-acetate- induced internalization of CD4 in a manner that depends on a physical association between p56(lck) and CD4. This decreased rate is due at least in part to a requirement for disruption of the CD4-p56(lck) complex prior to internalization of CD4. Furthermore, disruption of the CD4-p56(lck) complex appears to depend on the integrity of the cytoplasmic-domain serine at position 408, probably due to a requirement for phosphorylation.