Rat hepatocytes have previously been reported to possess prostaglandin E2 receptors of the EP3-type (EP3-receptors) that inhibit glucagon-stimulated glycogenolysis by decreasing cAMP. Here, the isolation of a functional EP3 beta receptor cDNA clone from a rat hepatocyte cDNA library is reported. This clone can be translated into a 362-amino-acid protein, that displays over 95% homology to the EP3 beta receptor from mouse mastocytoma. The amino- and carboxy-terminal region of the protein are least conserved. Transiently transfected HEK 293 cells expressed a single binding site for PGE2 with an apparent Kd of 15 nM. PGE2 > PGF2 alpha > PGD2 competed for [3H]PGE2 binding sites as did the EP3 receptor agonists M&B 28767 = sulprostone > misoprostol but not the EP1 receptor antagonist SC 19220. In stably transfected CHO cells M&B 28767 > sulprostone = PGE2 > misoprostol > PGF2 alpha inhibited the forskolin-elicited cAMP formation. Thus, the characteristics of the EP3 beta receptor of rat hepatocytes closely resemble those of the EP3 beta receptor of mouse mastocytoma.