We have examined the effects of adenosine receptors and protein kinases A and C in the regulation of erythropoietin production using hepatocellular carcinoma (Hep3B) cells in culture under normoxic (20% oxygen) and hypoxic (1% oxygen) conditions in vitro and in vivo in normal mice exposed to hypobaric hypoxia (.42 atm). CGS 21680, a selective adenosine A_2A agonist, significantly increased levels of erythropoietin in Hep3B cell cultures under normoxic (20% oxygen) conditions and in plasma of normal mice under both normoxic and hypoxic (4 hr; 0.42 atm) conditions. CGS 21680 also produced a significant increase in Epo mRNA levels in Hep3B cell cultures under normoxic conditions. SCH 58261, a selective adenosine A_2A receptor antagonist, significantly inhibited the increase in medium levels of Epo in Hep3B cell cultures exposed to hypoxia (1% O₂). Enprofylline, a selective adenosine A_2B receptor antagonist, significantly inhibited the increase in plasma levels of erythropoietin in normal mice following exposure to 2 and 4 hrs hypoxia. Chelerythrine chloride, an antagonist of protein kinase C activation, significantly inhibited hypoxia-induced increases in serum levels of erythropoietin in normal mice. A model is presented for hypoxic regulation of erythropoietin production which postulates that hypoxia causes increased release of ATP from cells into extracellular fluid (ECF) and increased ectonucleotidase activity resulting in increased ECF levels of adenosine. Adenosine activation of the A_2A receptors causes an increase in cyclic AMP, activation of kinase A and phosphorylation of an Epo transcriptional protein HIF1 which results in an increase in Epo mRNA. Adenosine also causes the activation of the A_2B receptors leading to the stimulation of phospholipase C which increases IP₃ and diacylglycerol (DAG). IP₃ causes mobilization of calcium from the endoplasmic reticulum and acts in concert with diacylglycerol to activate protein kinase C. Adenosine also acts on a receptor which activates the phospholipase A₂ pathway, leading to an increase in Cis unsaturated free fatty acids (CUFFA). DAG plus calcium and CUFFA activate PKC, leading to an increase in other transcriptional proteins which are involved in Epo gene expression.