The major site of phosphorylation of the epidermal growth factor (EGF) receptor after treatment of cells with EGF is threonine 669. Phosphorylation of this site is also associated with the transmodulation of the EGF receptor caused by platelet-derived growth factor and phorbol ester. A distinctive feature of the primary sequence surrounding threonine 669 is the proximity of 2 proline residues (-Pro-Leu-Thr669-Pro-). This site is not a substrate for phosphorylation by protein kinase C. To investigate the mechanism of the increased phosphorylation of the EGF receptor at threonine 669, in vitro assays were used to measure protein kinase and protein phosphatase activities present in homogenates prepared from cells treated with and without EGF. No evidence for the regulation of protein phosphatase activity was obtained in experiments using the [32P]phosphate-labeled EGF receptor as a substrate. A synthetic peptide corresponding to residues 663-681 of the EGF receptor was used as a substrate for protein kinase assays. Incubation of murine 3T3 L1 pre-adipocytes and human WI-38 fibroblasts with EGF caused a rapid increase (3-10-fold) in the level of threonine protein kinase activity detected in cell homogenates. Similar results were obtained after EGF treatment of Chinese hamster ovary cells expressing wild-type (Thr669) and mutated (Ala669) human EGF receptors. Activation of the threonine protein kinase activity was also observed in cells treated with platelet-derived growth factor, serum, and phorbol ester. Insulin-like growth factor-1 caused no significant change in protein kinase activity. Together these data indicate a role for the regulation of the activity of a threonine protein kinase in the control of the phosphorylation state of the EGF receptor at threonine 669. The significance of the identification of a growth factor-stimulated threonine protein kinase to the mechanism of signal transduction is discussed.