Background-Prostaglandin E2 (PGE(2)) EP receptors EP3 and EP4 signal via decreased and increased cAMP production, respectively. Previously, we reported that cardiomyocyte-specific EP4 knockout mice develop dilated cardiomyopathy with reduced ejection fraction. Thus, we hypothesized that PGE(2) increases contractility via EP4 but decreases contractility via EP3. Methods and Results-The effects of PGE(2) and the EP1/EP3 agonist sulprostone on contractility were examined in the mouse Langendorff preparation and in adult mouse cardiomyocytes. Isolated hearts of adult male C57Bl/6 mice were perfused with PGE(2) (10(-6) M) or sulprostone (10-6 M) and compared with vehicle. Both PGE(2) and sulprostone decreased + dp/dt (P<0.01) and left ventricular developed pressure (P<0.001) with reversal by an EP3 antagonist. In contrast, the EP4 agonist had the opposite effect. Adult mouse cardiomyocytes contractility was also reduced after treatment with either PGE(2) or sulprostone for 10 minutes. We then examined the acute effects of PGE(2), sulprostone, and the EP4 agonist on expression of phosphorylated phospholamban and sarcoendoplasmic reticulum Ca2+-ATPase 2a in adult mouse cardiomyocytes using Western blot. Treatment with either PGE(2) or sulprostone decreased expression of phosphorylated phospholamban corrected to total phospholamban, whereas treatment with the EP4 agonist had the opposite effect. Sarcoendoplasmic reticulum Ca2+-ATPase 2a expression was unaffected. Finally, we examined the effect of these compounds in vivo using pressure-volume loops. Both PGE(2) and sulprostone decreased + dp/dt, whereas the EP4 agonist increased + dp/dt. Conclusions-Contractility is reduced via the EP3 receptor but increased via EP4. These effects may be mediated through changes in phospholamban phosphorylation and has relevance to detrimental effects of inflammation.