Prostaglandin E(2) (PGE(2)) is a lipid mediator that acts by ligating 4 distinct G protein-coupled receptors, E prostanoid (EP) 1 to 4. Previous studies identified the importance of PGE(2) in regulating macrophage functions, but little is known about its effect on macrophage maturation. Macrophage maturation was studied in vitro in bone marrow cell cultures, and in vivo in a model of peritonitis. EP2 was the most abundant PGE(2) receptor expressed by bone marrow cells, and its expression further increased during macrophage maturation. EP2-deficient (EP2(-/-)) macrophages exhibited enhanced in vitro maturation compared with wild-type cells, as evidenced by higher F4/80 expression. An EP2 antagonist also increased maturation. In the peritonitis model, EP2(-/-) mice exhibited a higher percentage of F4/80(high)/CD11b(high) cells and greater expression of macrophage colony-stimulating factor receptor (M-CSFR) in both the blood and the peritoneal cavity. Subcutaneous injection of the PGE(2) analog misoprostol decreased M-CSFR expression in bone marrow cells and reduced the number of peritoneal macrophages in wild-type mice but not EP2(-/-) mice. The suppressive effect of EP2 ligation on in vitro macrophage maturation was mimicked by a selective protein kinase A agonist. Our findings reveal a novel role for PGE(2)/EP2/protein kinase A signaling in the suppression of macrophage maturation.