We examined the signal transduction of mouse prostaglandin E receptor EP1 subtype using Chinese hamster ovary cells stably expressing the cloned EP1. Sulprostone, an EP1 agonist, induced a rapid increase in intracellular Ca²⁺ concentration in the EP1-expressing cells. Most of the increase was abolished by removal of extracellular Ca²⁺, and was insensitive to U-73122, a phospholipase C inhibitor. Sulprostone stimulated phosphatidylinositol hydrolysis, but this stimulation was abolished by removal of extracellular Ca²⁺, indicating that EP1-stimulated phosphatidylinositol hydrolysis is the result of extracellular Ca²⁺ influx. Thus, the signal transduction of EP1 is extracellular Ca²⁺ entry through a pathway independent of phospholipase C activation. We further examined the regulation of the signal transduction of EP1 having potential phosphorylation sites for either protein kinase C or protein kinase A. Short-term exposure of the cells to 12-O-tetradecanoylphorbol 13-acetate (TPA) completely suppressed the sulprostone-induced increase in intracellular Ca²⁺- concentration, while forskolin or dibutyryl CAMP did not affect it, suggesting that protein kinase C but not protein kinase A is involved in the regulation of the EP1 signal transduction. Furthermore, long-term exposure to TPA decreased PGE₂ binding activity of EP1 due to the reduction of the EPI mRNA level. Protein kinase C induces short- and long-term desensitization of EP1.