Incubation of membranes prepared from A431 cells with either epidermal growth factor (EGF) or phorbol 12-myristate 13-acetate (PMA) stimulates the transfer of 32phosphate from [gamma-32P]ATP into 8-10 membrane proteins. The major phosphorylated protein migrates on NaDodSO4/polyacrylamide gels with an apparent Mr of 180,000, corresponding to the previously identified EGF receptor. Stimulation of EGF receptor phosphorylation by PMA does not require Ca2+, suggesting that prior activation of protein kinase C is not a prerequisite for phosphate transfer. PMA-enhanced phosphorylation proceeds at 4 degrees C and requires Mn2+, both properties of tyrosine-specific protein kinases. Phospho amino acid analysis of the Mr 180,000 receptor band shows that only tyrosine residues are phosphorylated when A431 membranes are treated with either EGF or PMA. Moreover, proteolysis reveals that these residues are located in the same peptides of the receptor. These results demonstrate that a potent tumor-promoting phorbol ester can mimic a critical early response usually elicited by EGF.