We have previously demonstrated that mRNAs for erythropoietin and the erythropoietin receptor temporarily express on the visceral yolk sacs on days 9-11 of gestation in mice. In order to investigate the sites of expression, we performed in situ hybridization on visceral yolk sacs. Visceral yolk sacs from 10-day-old mice embryos were frozen in liquid nitrogen, and processed for cryosections. Sections were hybridized with a 35S-labeled RNA probe complementary to mRNA coding for erythropoietin or erythropoietin receptor. Erythropoietin mRNA was detectable in 57.6% of the endodermal epithelial cells, while erythropoietin-receptor mRNA was discerned in 90.8% of the endodermal cells and mesodermal cells, including hemocyteblasts. Moreover, erythropoietin protein was detectable in 52.8% of the endodermal epithelial cells, and on the surface of hemocyteblasts and mesothelial cells. Erythropoietin-receptor protein was discernible in 87.2% of the endodermal cells and in the corresponding mesodermal cells to those where erythropoietin protein was expressed by immunohistochemical examinations. The results indicate that erythropoietin-synthesizing cells are located in half of the endodermal epithelial cells, while the majority of cells in the visceral yolk sac are erythropoietin-receptor-producing cells, indicating that almost all cell population in the visceral yolk sac is erythropoietin-responding cells via both autocrine and paracrine routes.