We compared the direct effects of selective EP4 and EP2 receptor agonists (EP4A and EP2A) with prostaglandin E(2) (PGE(2)) on the differentiation of cultured murine calvarial osteoblastic cells. EP4A increased alkaline phosphatase activity and osteocalcin mRNA levels in these cultures similar to PGE(2). This effect was seen with both "direct plating" immediately after isolating the cells, or "indirect plating" in which the cells were grown to confluence and replated. EP2A had a smaller effect, significant only in "indirect plating" experiments. All three agents decreased the DNA and protein content in indirect plating experiments, but not in direct plating experiments. We conclude that the anabolic effect of PGE(2) in calvarial osteoblastic cell cultures is largely mediated by activation of the EP4 receptor, while activation of the EP2 receptor is less effective.