Prostaglandin E2 (PGE2) is an anabolic agent in vivo that stimulates bone formation by recruiting osteoblasts from bone marrow precursors. To understand which of the known PGE2 receptors (EP1-4) is involved in this process, we tested the effect of PGE2 and various EP agonists and/or antagonists on osteoblastic differentiation in cultures of bone marrow cells by counting bone nodules and measuring alkaline phosphatase activity. PGE2 increased both parameters, peaking at 100 nM, an effect that was mimicked by forskolin and was abolished by 2',3'-dideoxyadenosine (an adenylate cyclase inhibitor) and was thus cAMP dependent, pointing to the involvement of EP2 or EP4. Consistently, 17-phenyl-omega-trinor PGE2 (EP1 agonist) and sulprostone (EP3/EP1 agonist) lacked any anabolic activity. Furthermore, butaprost (EP2 agonist) was inactive, 11-deoxy-PGE1 (EP4/EP2 agonist) was as effective as PGE2, and the PGE2 effect was abolished dose dependently by the selective EP4 antagonist AH-23848B, suggesting the involvement of EP4. We also found that PGE2 increased nodule formation and AP activity when added for the initial attachment period of 24 h only. Thus this study shows that PGE2 stimulates osteoblastic differentiation in bone marrow cultures, probably by activating the EP4 receptor, and that this effect may involve recruitment of noncommitted (nonadherent) osteogenic precursors, in agreement with its suggested mode of operation in vivo.