Erythropoietin is a glycoprotein factor which specifically regulates the proliferation and differentiation of erythroid progenitor cells. We have investigated here the biochemical mechanisms of erythroid differentiation on mouse erythroleukemia SKT6 cells which can be induced to differentiate either with erythropoietin or dimethyl sulfoxide (Me,SO). CAMP-elevating agents, such as forskolin and 3-isobutyl-l-methyl-xanthine, caused spontaneous erythroid differentiation, and these agents showed the stimulatory effects on erythropoietin-or Me2SO-induced differentiation. An adenylate cyclase inhibitor, 2',5'-dideoxyadenosine, blocked erythropoietin-induced differentiation. The intracellular cAMP level was rapidly increased by addition of erythropoietin but not by Me2S0. These observations suggest that erythroid differentiation induced by erythropoietin is mediated, at least in part, through the CAMP-dependent pathway. When the effect of erythropoietin and Me2S0 on the intracellular Ca2+ level was examined using fura 2, no acute change was observed. Measurements of the levels of inositol1,4,5-trisphosphate and diacylglycerol following stimulation with erythropoietin or Me2S0 showed that phosphatidylinositol turnover did not change significantly after erythropoietin stimulation but decreased gradually after MezSO induction. Taken together, these results indicate that a complex signaling network including the CAMP-dependent pathway is involved in the erythroid differentiation process.Hemopoiesis is regulated by a set of specific glycoprotein factors that affect the program for proliferation and differentiation of progenitor cells [l]. Among these humoral factors, three glycoproteins specific for erythropoietic lineage have been identified by recombinant DNA technology, and their physiological functions have been clarified [2 -61. Interleukin 3 [2] and granulocyte-macrophage colony-stimulation factor [3, 41 stimulate the proliferation and differentiation of early erythroid progenitor cells. Erythropoietin acts on more mature erythroid progenitor cells and induces differentiation of the cells to mature erythrocytes [5 -71. Erythropoietin has been purified to homogeneity from the urine of a patient with aplastic anemia [7]. Clones of complementary and genomic DNA encoding the human and mouse erythropoietin were isolated [5,6,8, 91 and the primary structures of the protein and its sugar moieties have been determined [ 101.Study of the action of this hormone has been hampered partly by the lack of a sufficient number of homogeneous cells which fully respond to erythropoietin. Recently we established a mouse erythroleukemia cell line SKT6 which can be induced to differentiate with either natural inducer erythropoietin or Correspondence to K. Todokoro,