Because global deletion of the prostaglandin EP4 receptor results in neonatal lethality, we generated a mouse with targeted EP4 receptor deletion using Cre–LoxP methodology and a 2.3 kb collagen I a1 promoter driving Cre recombinase that is selective for osteoblastic cells. We compared wild type (WT), global heterozygote (G-HET), targeted heterozygote (T-HET) and knockout (KO) mice. KO mice had one targeted and one global deletion of the EP4 receptor. All mice were in a mixed background of C57BL/6 and CD-1. Although there were one third fewer G-HET or KO mice at weaning compared to WT and T-HET mice, G-HET and KO mice appeared healthy. In cultures of calvarial osteoblasts, prostaglandin E₂ (PGE₂) increased alkaline phosphatase (ALP) activity in cells from WT mice, and this effect was significantly decreased in cells from either G-HET or T-HET mice and further decreased in cells from KO mice. A selective agonist for EP4 receptor increased ALP activity and osteocalcin mRNA levels in cells from WT but not KO mice. A selective COX-2 inhibitor, NS-398, decreased osteoblast differentiation in WT but not KO cells. At 15 to 18 months of age there were no differences in serum creatinine, calcium, PTH, body weight or bone mineral density among the different genotypes. Static and dynamic histomorphometry showed no consistent changes in bone volume or bone formation. We conclude that expression of the EP4 receptor in osteoblasts is critical for anabolic responses to PGE₂ in cell culture but may not be essential for maintenance of bone remodeling in vivo.