Epidermal growth factor (EGF)-enhanced protein kinase activity in plasma membrane preparations of A-431 human epidermoid carcinoma cells was shown to involve the phosphorylation of tyrosine residues. Phosphotyrosine was detected in both endogenous membrane proteins and in histone when added as an exogenous protein substrate. The major phosphorylated amino acid in partial acid hydrolysates of 32P-labeled A-431 membranes was identified as phosphotyrosine on the basis of its identical behavior to authentic phosphotyrosine on paper electrophoresis and thin layer chromatography; its 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) derivative was indistinguishable from that of the authentic compound. Only traces, if any, of phosphoserine or phosphothreonine were detected. [32P]Phosphotyrosine was also detected in pronase digests of 32P-labeled membrane proteins. The EGF receptor . protein kinase complex, which was solubilized with Triton X-100 and purified by EGF affinity chromatography, was shown to phosphorylate tyrosine residues of the isolated membrane protein.