We have shown that a type I phosphatidylinositol (PI) kinase activity is associated with the epidermal growth factor (EGF) receptor in a mouse fibroblast cell line expressing human EGF receptors (NRHER5) and that this activity increases dramatically upon treatment of cells with physiologically relevant concentrations of EGF. EGF stimulated a time-dependent increase in EGF receptor-associated PI kinase activity measured in EGF receptor immunoprecipitates. Activation was detected 15 min after the addition of EGF, and it peaked between 1 and 2 hr. Activation of PI kinase was detected with EGF concentrations as low as 10 pM and maximal stimulation occurred at approximately 1 nM. Analysis of deacylated PI phosphate products, and inhibition of the PI kinase activity by nonionic detergent, indicated that the PI kinase described here was type I or PI 3' kinase. These results demonstrate the regulation of a type I PI kinase by EGF and suggest a potential role in the EGF receptor signal transduction pathway.