A functional cDNA clone for the mouse prostaglandin (PG) E receptor EP₂ subtype was isolated from a mouse cDNA library. The mouse EP₂ receptor consists of 362 amino acid residues with seven putative transmembrane domains. [³H]PGE₂ bound specifically to the membrane of Chinese hamster ovary cells stably expressing the cloned receptor. This binding was displaced by unlabeled prostanoids in the order of iloprost, a stable PGI₂ agonist > PGF_2α > PGD₂. Binding was also inhibited by butaprost (an EP₂ agonist) and to a lesser extent by M&B 28767 (an EP₃ agonist), but not by sulprostone (an EP₁ and EP₃ agonist) or SC‐19220 (an EP₁ antagonist). PGE₂ and butaprost increased the cAMP level in the Chinese hamster ovary cells in a concentration‐dependent manner. Northern blot analysis revealed that EP₂ mRNA is expressed most abundantly in the uterus, followed by the spleen, lung, thymus, ileum, liver, and stomach.