To assess the functional activity of internalized epidermal growth factor (EGF) receptors in A-431 cells we investigated their ability to be both the target and activator of serine and threonine protein kinases. By incubating A-431 cells with EGF at 4 degrees C wr at 37 degrees C, eluting surface-bound EGF with an acid buffer, and immunoprecipitating the EGF receptor with different antibodies, we were able to compare the phosphorylation state of internalized EGF receptors to those found on the plasma membrane in intact cells. Tryptic phosphopeptide mapping and subsequent phosphoamino acid analysis revealed four tyrosine, one threonine, and seven serine phosphorylation sites in the molecule of plasma membrane receptor, while internalized EGF receptor contained one additional threonine and three additional serine phosphorylation sites. Because acid-mediated removal of EGF from its receptor demonstrated that the majority of EGF-induced phosphorylation required the continuous presence of activated receptor to be maintained, the conclusion was made that internalized EGF receptors may be not only the target of protein kinases whose activity was detected in our assay but also an activator of at least some of them. The interaction between internalized EGF receptors and nontyrosine protein kinase was also observed in vitro. Membrane-associated protein kinase was detected which phosphorylated a serine residue of the EGF receptor molecule in an EGF-dependent manner. Subcellular fractionation revealed the presence of the serine protein kinase both in the fractions of plasma membranes and high-density endosomes. These results demonstrate that at its prelysosomal stage, internalization does not impair the functional activity of EGF receptor.