Epidermal growth factor (EGF) stimulates the phosphorylation by A431 membranes of tyrosine residues in the myosin regulatory light chain (LC20) from chicken gizzard smooth muscle. In the presence of EGF, the Km of the EGF receptor kinase for LC20 is 73 microM and the V max is 17 nmol/min/mg. Two moles of phosphate are incorporated into tyrosine per mol of LC20. Trypsin digestion of the phosphorylated LC20 produces two phosphopeptides which are phosphorylated to approximately equal extents. Sequential Edman degradation of the separated phosphopeptides shows that tyrosines 142 and 155 of the protein are phosphorylated. Tyrosine 142 is located within a sequence similar to that of autophosphorylated tyrosine kinases in that five of the seven amino acids on the NH2-terminal side are acidic. Tyrosine 155 has no acidic amino acids near its NH2-terminal side. A comparison of the initial rates of phosphorylation of the two tyrosines shows that tyrosine 142 is phosphorylated at three times the rate of tyrosine 155.