We have identified the sites phosphorylated in vitro by epidermal growth factor (EGF) receptor kinase in bovine brain phospholipase C-gamma (PLC-gamma). They are tyrosine residues 472, 771, 783, and 1254. The rate of phosphorylation was fastest with the sites at 771 and 783, then at 1254, and slowest at 472. PLC-gamma isolated from cells treated with EGF is known to contain at least four tyrosine phosphate-containing peptides and two of them are identified to be residues 771 and 1254 in the accompanying paper (Wahl, M. I., Nishibe, S., Kim, J. W., Kim, H., Rhee, S. G., and Carpenter, G. (1990) J. Biol. Chem. 265, 3944-3948). The 3 residues 472, 771, and 783 are located closely to the regions of PLC-gamma which exhibit a high sequence similarity to the regulatory domain of the src family tyrosine kinases. Nevertheless, the tyrosine phosphorylation did not affect the catalytic activity of PLC-gamma in vitro. We propose, therefore, that the phosphorylation of PLC-gamma by EGF receptor kinase alters its interaction with putative inhibitory proteins and leads to its activation.