The EP3 receptor for prostaglandin E2 (PGE2) mediates various biological activities such as uterine contraction, inhibition of gastric acid secretion, presynaptic inhibition of neurotransmitter release and potentiation of platelet aggregation. In an attempt to understand the molecular basis of this diversity of biological function, we cloned full-length cDNAs encoding EP3 receptors for PGE2 from human uterus cDNA libraries. Seven cDNA variants were identified which code for six distinct EP3-receptor isoforms. Sequencing revealed that the receptor isoforms differ in their intracellular C-terminal domains. Southern blot experiments indicate that the isoforms are generated by alternative splicing. The EP3-receptor gene is expressed in various tissues with high expression in kidney and pancreas, as demonstrated by Northern blot analysis. All receptors, stably expressed in baby hamster kidney (BHK) cells, bind PGE2 specifically with similar Kd of 2.2-5.8 nM. The binding of [3H]PGE2 is competed with by unlabelled prostaglandins in the order sulprostone (a PGE2-like agonist) approximately PGE2 >> PGF2 alpha > Iloprost (a prostacyclin analogue) > PGD2, which is specific for EP3 receptors. Analysis of the signal-transduction pathways demonstrated that all receptors respond with inhibition of forskolin-induced cAMP accumulation with an IC50 of 0.1-3 nM PGE2. In addition, some isoforms induce an increase in intracellular free calcium ([Ca2+]i) at PGE2 concentrations greater than or equal to 10 nM. These results may offer an explanation for the different physiological responses observed in various tissues following activation of EP3 receptors.