Erythropoietin (Epo) is the main regulator of erythrocyte production and a potent cytoprotective factor. It was suggested that some of Epo cytoprotective properties are due to its regulation of nitric oxide (NO) production. Recently, functionally active endothelial type NO synthase (eNOS) was discovered in mature murine and human red blood cells (RBC-eNOS). The goal of the present study was to characterize the effect of physiological and therapeutic doses of Epo on RBC-eNOS function. We found that recombinant human Epo (rHuEpo) binds specifically to mouse erythrocytes. Epo binding sites are not equally distributed through the RBC population but prevail in reticulocytes and young erythrocytes with about 105 receptors/cell, compared with adult and old erythrocytes containing 1-4 receptors/cell. The treatment of mouse erythrocytes with rHuEpo resulted in a time- and dose-dependent upregulation of NO production mediated via activation of the phosphatidylinositol-3-kinase /Akt pathway and RBC-eNOS phosphorylation at Ser-1177. Finally, when erythrocytes were incubated in L-arginine-free medium, rHuEpo treatment resulted in upregulation of superoxide radical production with concomitant shifting of the cellular redox state toward more oxidized state. Epo-induced changes in erythrocyte redox potential were absent in erythrocytes from eNOS-deficient mice.