Ligand-activated epidermal growth factor (EGF) receptors are coupled to the phosphatidylinositol (PtdIns) pathway to stimulate formation of two second messengers, inositol trisphosphate and diacylglycerol. Investigation of the interaction between EGF receptors and phosphoinositide kinases identified PtdIns and PtdIns(4)P kinase activities in extensively washed EGF receptor immunoprecipitates. Studies using COOH-terminal truncation mutant EGF receptors and immunoisolation by an EGF receptor peptide anti-serum in the presence of peptide (residues 644-666) indicated that the phosphoinositide kinases were associated with the region located between the inner membrane boundary and the kinase domain of the EGF receptor. In vivo cross-linking identified four tyrosine phosphorylated proteins of approximately 135, 62, 55, and 47 kDa associated with the EGF receptor. After EGF stimulation, PtdIns and PtdIns(4)P kinase activities were markedly increased among proteins isolated by monoclonal antiphosphotyrosine antibodies. The activities associated with the EGF receptor and with tyrosine-phosphorylated proteins were identified as PtdIns4-and PtdIns(4)P 5-kinase. Tyrosine dephosphorylation did not alter the activity of the prominent PtdIns(4)P 5-kinase activity. These results indicate that the phosphoinositide kinases are associated with and tyrosine phosphorylated by the EGF receptor as part of the mechanism coordinating responses between signal transduction pathways but do not demonstrate that tyrosine phosphorylation of PtdIns(4)P 5-kinase is sufficient to activate the enzyme.