Prostaglandin E₂ (PGE₂) stimulates osteoclast formation by increasing receptor activator of nuclear factor (NF)-κB ligand (RANKL) mRNA expression via cAMP-protein kinase A (PKA) pathways in osteoblasts. Since phosphodiesterases (PDEs) balance the concentration of intracellular cAMP stimulated by PGE₂, we investigated the role of PDEs in PGE₂-mediated osteoclast formation using various cAMP-specific PDEs inhibitors. In the presence of PGE₂, PDE3 and 4 inhibitors were shown to dose-dependently increase the osteoclast formation in cocultures of mouse bone marrow cells and calvarial osteoblasts. In agreement with this finding, they stimulated PGE₂-induced cAMP production followed by increased RANKL mRNA expression in osteoblasts, suggesting that PDE3 and 4 negatively regulate PGE₂-mediated RANKL expression in osteoblasts. RT-PCR analysis revealed that PDE3A, 3B, 4A, 4B and 4D are expressed in osteoblasts. The PDE8 inhibitor did not increase osteoclast formation, although it stimulated PGE₂-induced RANKL mRNA expression in osteoblasts. The four subtypes of PGE receptors are designated EP1, EP2, EP3, and EP4. PDE3 and 4 inhibitors were found to increase EP1/3, EP4 and/or EP2 agonist-stimulated RANKL expression, indicating that PDE3 and PDE4 negatively regulate PGE₂-induced RANKL mRNA expression through four EPs. Taken together, these data suggest that PDE3 and PDE4 could have important pharmacological and clinical implications in bone-related diseases.