The phosphorylation of a prominent 43-kDa phosphoprotein (pp43) in the membranes of normal murine erythroid cells was reduced markedly by exposure of the membranes to highly purified erythropoietin. A virtually identical reduction of pp43 phosphorylation was seen when erythropoietin-responsive Rauscher murine erythroleukemia cell membranes were exposed to the hormone. This effect was both time-dependent, occurring within 30 min after erythropoietin exposure, and concentration-dependent. Phosphoamino acid analysis revealed that pp43 is phosphorylated on serine residues. The results provide the first evidence that rapid alterations in membrane protein phosphorylation may serve as a trans-membrane signal for erythropoietin.