The protein of the Raf-a proto-oncogene is a protein serine/threonine kinase that is activated after stimulation of cells with insulin, epidermal growth factor, and other mitogens. Incubation with these mitogens leads to a shift in electrophoretic mobility, which is caused by an increased phosphate incorporation into the Raf-I protein on serine and threonine residues. To investigate the mechanism of this activation, we used purified Raf-I expressed in Escherichia coli as a substrate for a putative Raf-1 protein kinase. In three different insulin-sensitive cell types, insulin-activated Raf-I kinase was localized in the crude cytosolic fractions. The insulin-stimulated Raf-I kinase kinase(s) phos- phorylated the purified Raf-I protein on multiple sites in vitro, as evidenced by tryptic peptide mapping. At least some of these phosphorylation sites appear to overlap with sites phosphorylated in response to serum in intact cells. The insulin-stimulated Raf-I kinase kinase may play a role in mediating the phosphorylation and possibly the activation of the Raf-I kinase by insulin and other growth factors. Enzymatically active Raf-1 kinase was partially purified from baculovirus-expressed Raf-I protein. This enables us to determine the activating phosphorylation site of the Raf-1 kinase and to search for in vitro substrates. Receptor-mediated activation of Raf-1 protein kinase is followed by translocation of the normally-cytosolic enzyme to the perinuclear area and the nucleus. Raf-I dependence of growth induction by three hematopoietic growth factors that we recently showed to regulate Raf-1 activity was tested with antisense oligonucleotides. All three receptors require Raf-I for induction of DNA synthesis.