We have examined the phosphorylation of the serine threonine kinase, the product of c-raf proto-oncogene in response to insulin or platelet-derived growth factor in intact cells. Both insulin and platelet-derived growth factor stimulated phosphorylation of the c-raf protein about 2- to 3-fold. The phosphorylation occurred exclusively on serine and threonine residues; phosphotyrosine was not detected. In immune-complex kinase assays, treatment with insulin, and platelet-derived growth factor increased autophosphorylation of the c-raf kinase, suggesting activation of its kinase activity. To investigate whether the phosphorylation of the c-raf protein in intact cells results from an autophosphorylation event or from the phosphorylation by other cellular kinase(s), we replaced lysine 375 in the putative ATP-binding domain of the c-raf protein with alanine using oligonucleotide site-directed mutagenesis and expressed the mutated protein in NIH3T3 cells. The substitution resulted in the inactivation of the serine/threonine-specific autophosphorylation in immune-complex kinase assays. In intact cells, however, although phosphorylation of the mutant protein in response to insulin and platelet-derived growth factor occurred to a lesser extent than that of the wild-type protein, the phosphopeptide maps were indistinguishable. These results suggest that serine threonine phosphorylation might be responsible for the activation of c-raf kinase upon treatment of cells with insulin and platelet-derived growth factor, and most of the phosphate associated with the c-raf protein results from its phosphorylation by as yet uncharacterized cellular serine/threonine kinase(s).