Rat hepatocytes have previously been reported to possess prostaglandin E, receptors of the EP,-type (EP,-receptors) that inhibit glucagonstimulated glycogenolysis by decreasing CAMP. Here, the isolation of a functional EP, 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 EP, 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 PGE, with an apparent Kd of 15 nM. PGE, > PGF,, > PGDz competed for ['HIPGEl binding sites as did the EP, receptor agonists M&B 28767 = sulprostone > misoprostol but not the EP, receptor antagonist SC 19220. In stably transfected CHO cells M&B 28767 > sulprostone = PGE, > misoprostol > PGF, inhibited the forskolin-elicited CAMP formation. Thus, the characteristics of the EP, receptor of rat hepatocytes closely resemble those of the EP, receptor of mouse mastocytoma.