The phorbol ester phorbol myristate acetate (PMA) induces a rapid downregulation of CD4 from the surface of T cells and lymphocytic cell lines, as well as from CD4-transfected nonlymphoid cells. Here we have studied the mechanisms of this phorbol ester-induced CD4 modulation. Using HeLa-CD4 or NIH-3T3-CD4 cells, in which the endocytosis of CD4 is not influenced by the protein tyrosine kinase p56lck, we show that PMA enhanced the uptake of CD4, increasing the rate of CD4 endocytosis three to five-fold, and doubling the proportion of CD4 found inside the cells. Trafficking of a CD4 mutant lacking the major portion of the cytoplasmic domain, as well as fluid phase endocytosis were not affected by PMA treatment. Studies in which clathrin-coated pits were disrupted through the use of hypertonic media indicated that both the constitutive and PMA-induced CD4 uptake occurred through coated vesicles. Electron microscopy demonstrated directly that PMA increases the association of CD4 with coated pits. Immunofluorescent staining of internalized CD4 showed that PMA also diverted CD4 from the early endosome-plasma membrane recycling pathway to a mannose 6-phosphate receptor-containing late endosomal compartment. In lymphoid or p56lck-expressing transfected cells, these effects were preceded by the PMA-induced dissociation of CD4 and p56lck, which released CD4 and made possible increased endocytosis and altered intracellular trafficking. Together these results indicate that phorbol esters have multiple effects on the normal endocytosis and trafficking of CD4, and suggest that phosphorylation may influence the interaction of CD4 with coated pits.