PGE2 is one of the key molecules in the osteoblast. It is the major prostanoid in the bone, and its production is under the control of both systemic and local factors. PGE2 has been reported to have multiple actions in the osteoblast, such as growth promotion and cell differentiation. To better understand the action of PGE2 in the osteoblast, we determined the PGE receptor subtypes in MC3T3-E1, an osteoblastic cell line derived from the normal mouse calvaria. Northern blot analysis revealed that EP1 and EP4 subtypes are expressed in MC3T3-E1. In contrast, EP3 subtype was not detected by either Northern blot analysis or RT-PCR. The contribution of each subtype was evaluated by studying the effects of subtype-specific analogs on osteoblastic function at confluency and 5 days after confluency. An EP1 agonist, 17-phenyl-omega-trinor PGE2, increased DNA synthesis and decreased alkaline phosphatase activity. 11-Deoxy-PGE1, and EP2 and EP4 agonist, decreased DNA synthesis and increased alkaline phosphatase activity at both stages. Butaprost, an EP2-selective agonist, showed effects similar to those of 11-deoxy-PGE1 only at confluency. Another and more differentiated osteoblastic marker, osteocalcin production, was detectable and was stimulated by 11-deoxy-PGE1 only 5 days after confluency. The exposure of these cells to EP1 agonist changed the cell shape to a more fibroblastic appearance. These results indicate that EP1, EP4, and probably EP2 are present in MC3T3-E1 cells; EP1 promotes cell growth, and EP2 and EP4 mediate differentiation of the osteoblast. Furthermore, the decreased response to EP2-specific agonist 5 days after confluency suggests that the expression of PGE receptor subtype is dependent on the stage of osteoblastic differentiation. This is the first report to determine PGE receptor subtypes in the bone.