The proto-oncogene c-myc has been identified as an early response gene for erythropoietin (Epo) in transformed murine erythroleukemia cells. Epo activation of c-myc in these cells requires protein kinase C. We now show the fidelity of this signaling pathway in normal erythroid cells isolated from the spleens of phenylhydrazine-treated mice. Mouse spleen cells rich in erythroid progenitors were washed free of endogenous Epo and then incubated in the absence of Epo. Subsequent addition of Epo for 1 hour led to a dramatic elevation of c-myc transcript. Addition of the protein synthesis inhibitor cycloheximide did not prevent the c-myc response, thus identifying c-myc as an Epo early response gene in normal cells. We used this c-myc response as a reporter for signals initiated by the Epo receptor. Using a series of inhibitors with known specificities and established rank-orders of potency for different kinases, we determined that the c-myc response to Epo was blocked with the following rank order: staurosporine much greater than H7 greater than sangivamycin greater than H8. This sequence is identical to that obtained using transformed cells and is diagnostic of a protein kinase C-dependent signal. Because direct activation of protein kinase by phorbol esters does not induce terminal differentiation of normal cells, the pathway to c-myc established by these studies must represent one part of a signal transduction mechanism.