Erythropoietin, the prime regulator of red blood cell growth and differentiation, causes rapid changes in the phosphorylation of several integral plasma membrane proteins (Choi, H-S., Wojchowski, D. M., and Sytkowski, A. J. (1987) J. Biol. Chem. 262, 2933-2936; Choi, H-S., Bailey, S. C., Donahue, K. A., Vanasse, G. J., and Sytkowski, A. J. (1990) J. Biol. Chem. 265, 4143-4148). In the present study we have demonstrated that erythropoietin's signal is transduced rapidly to the cytosol resulting in specific phosphorylation/dephosphorylation events. Erythropoietin treatment of Rauscher murine erythroleukemia cells previously labeled with [32P]orthophosphate results in a rapid increase in phosphorylation of two cytosolic proteins, designated pp96 and pp80, and a decrease in phosphorylation of another protein, designated pp90. The relative molecular mass and pI of pp80 are virtually identical to those reported for the protein kinase C substrate p80, or "MARCKS protein." Treatment of the cells with 12-O-tetradecanoylphorbol-13-acetate also increases pp80 but not pp96 phosphorylation, suggesting that erythropoietin triggers a protein kinase C-dependent pathway to pp80 and a protein kinase C-independent pathway to pp96. The effect of erythropoietin on pp96 phosphorylation was also shown in nontransformed erythroid cells isolated from the spleens of phenylhydrazine-treated mice. In contrast, almost no 32P labeling of pp80 or pp90 was detected, and pp80 and pp90 protein were nearly absent from these normal cells. These differences in expression and phosphorylation of erythropoietin-sensitive phosphoproteins may be related to the growth factor independence or dependence of the erythroid cells.