We have characterized the rat prostanoid EP₁, EP₂, EP_3α and EP₄ receptor subtypes cloned from spleen, hepatocyte and/or kidney cDNA libraries. Comparison of the deduced amino acid sequences of the rat EP receptors with their respective homologues from mouse and human showed 91% to 98% and 82% to 89% identity, respectively. Radioreceptor binding assays and functional assays were performed on EP receptor expressing human embryonic kidney (HEK) 293 cells. The K_D values obtained with prostaglandin E₂ for the prostanoid receptor subtypes EP₁, EP₂, EP_3α and EP₄ were approximately 24, 5, 1 and 1 nM, respectively. The rank order of affinities for various prostanoids at the prostanoid receptor subtypes EP₂, EP_3α and EP₄ receptor subtypes was prostaglandin E₂=prostaglandin E₁>iloprost>prostaglandin F_2α>prostaglandin D₂>U46619. The rank order at the prostanoid EP₁ receptor was essentially the same except that iloprost had the highest affinity of the prostanoids tested. Of the selective ligands, butaprost was selective for prostanoid EP₂, M&B28767 and sulprostone were selective for EP_3α and enprostil displayed dual selectivity, interacting with both prostanoid receptor subtypes EP₁ and EP_3α. All four receptors coupled to their predominant signal transduction pathways in HEK 293 cells. Notably, using a novel aequorin luminescence assay to monitor prostanoid EP₁ mediated increases in intracellular calcium, both iloprost and sulprostone were identified as partial agonists. Finally, by Northern blot analysis EP₃ transcripts were most abundant in liver and kidney whereas prostanoid EP₂ receptor mRNA was expressed in spleen, lung and testis and prostanoid EP₁ receptor mRNA transcripts were predominantly expressed in the kidney. The rat prostanoid EP₁ probes also detected additional and abundant transcripts present in all the tissues examined. These were found to be related to the expression of a novel protein kinase gene and not the prostanoid EP₁ gene [Batshake, B., Sundelin, J., 1996. The mouse genes for the EP₁ prostanoid receptor and the novel protein kinase overlap. Biochem. Biophys. Res. Commun. 227, 1329–1333].