p56lck, a member of the src family of non-receptor protein tyrosine kinases (PTKs), is expressed predominantly in T-lymphocytes. Association of p56lck with CD4 and CD8 T-cell receptor (TcR) accessory molecules suggests that p56lck may play a specialized role in antigen-induced T-cell activation. CD4 and CD8 molecules are known to stabilize the interaction between TcR and the major histocompatibility complex during T-cell activation. To examine the role of p56lck in the dynamics of the CD4 molecule, p56lck-expressing transfectant cell clones were prepared by the transfection of an lck-gene plasmid containing an inducible promoter into a CD4+lck- human monocytoid cell line. When these transfectant cells were stimulated with phorbol ester, CD4 internalization on these p56lck-expressing cell lines was selectively and markedly retarded, as compared to p56lck-negative control cell lines. When cell-surface CD4 and intracellular CD4 were selectively precipitated after stimulation, the intracellular CD4 molecules were dissociated from p56lck whereas the surface-retained CD4 molecules were still associated with p56lck. Moreover, the dissociation of p56lck from CD4 appeared to occur prior to the PMA-induced internalization of CD4. These data indicate that p56lck regulates the PMA-induced internalization of CD4 possibly via its association with CD4. Treatment with genistein, a PTK inhibitor, revealed that the PTK activity of p56lck might not be involved in this regulatory effect of p56lck on CD4 internalization.