The effect of erythropoietin on the splenic microvascular system of polycythemic CF1 mice was studied using in vivo microscopic methods. Administration of a single dose (3 U) of erythropoietin resulted in an increase in the linear velocity of blood flow through the splenic sinusoids and a reduction in the number of sinusoids storing blood. This response was first seen 4-6 hr after injection; it persisted for 48 hr and was reduced markedly by 72 hr. By 120 hr the spleens were indistinguishable from controls. The response was specific for erythrogenic tissue, since no response was seen in the adjacent nonerythropoietic pancreatic tissue. The results suggest that the splenic microvascular response to erythropoietin may be indirect and may be mediated by the release of a vasoactive metabolite from the erythrogenic tissues surrounding the sinusoids. Erythropoietin-sensitive stem cells are suggested to be the source of such a metabolite.