Phosphorylation of the epidermal growth factor (EGF) receptors in endosomes isolated from A431 cells was studied using antiphosphotyrosin antibody (anti-P-Tyr). A431 cells were preincubated with EGF and then washed with acid buffer to remove surface-bound EGF. Endosomes were isolated from such cells by the method of subcellular fractionation on Percoll density gradient. Addition to isolated endosomes of anti-P-Tyr complexes with immunogold resulted in a significant shift of endosome peak to the high density region. This fact indicates that anti-P-Tyr interacts with phosphotyrosine residues of EGF receptors localized in endosomes.