We recently cloned the mouse prostaglandin (PG) E receptor EP₃ subtype that is coupled to adenylate cyclase inhibition through Gi and identified three isoforms which are produced through alternative splicing. In Chinese hamster ovary cells expressing each EP₃ isoform, PGE₂ induced an immediate increase in the intracellular Ca²⁺ concentration ([Ca²⁺]i) due to both Ca²⁺ mobilization from internal stores and influx from the extracellular medium. This increase was abolished by prior treatment with pertussis toxin (PT). PGE₂ also stimulated an accumulation of inositol trisphosphate (IP₃) in a PT-sensitive manner. Both the PGE₂-induced increase in [Ca²⁺]i and accumulation of IP₃ were blocked by the phospholipase C inhibitor U-73122. Thus, EP₃ is linked to phospholipase C activation via Gi, and this activation leads to Ca²⁺ mobilization from internal stores and influx from the extracellular medium.