The contention that erythropoietin (Epo) affects platelet production was investigated in the rat with recombinant human Epo (rHuEpo). In normal rats, Epo caused a dose-dependent increase in both reticulocyte and platelet numbers, the reticulocyte response preceding that of platelets. Withdrawal of Epo resulted in reticulocytes and platelets returning to control levels. [75Se]-selenomethionine incorporation into platelets was also enhanced in response to Epo. Chronic daily administration of rHuEpo resulted in steady state erythrocyte levels after 12 to 14 days, which were elevated 20% above controls. Attainment of this steady state was associated with both reticulocytes and platelets returning to control levels despite continued administration of Epo, an effect not associated with a change in the half-life of circulating Epo. In polycythemic rats a platelet response was observed before an effect on reticulocytes. Erythropoietin caused a 2.4-fold increase in the frequency of small acetylcholinesterase-positive cells within 24 hours, and increased the mean megakaryocyte diameter within 48 hours. Furthermore, the [3H]-thymidine labeling index of megakaryocytes from rats treated for 24 hours with rHuEpo was increased for all stages of megakaryocyte maturation. These results support the proposal of an effect of Epo on rat megakaryocytes causing increased platelet production.