Prostaglandin E₂ (PGE₂) stimulates the formation of osteoclast-like tartrate-resistant acid phosphatase-positive multinucleated cells (TRAP + MNC) in vitro. This effect likely results from stimulation of adenylyl cyclase, which is mediated by two PGE₂ receptors, designated EP₂ and EP₄. We used cells from mice in which the EP₂ receptor had been disrupted to test its role in the formation of TRAP + MNC. EP₂ heterozygous (±) mice in a C57BL/6 x 129/SvEv background were bred to produce homozygous null (EP₂ −/−) and wild-type (EP₂ +/+) mice. PGE₂, PTH, or 1,25 dihydroxyvitamin D increased TRAP+ MNC in 7-day cultures of bone marrow cells from EP₂ +/+ mice. In cultures from EP₂ −/− animals, responses to PGE₂, PTH, and 1,25 dihydroxyvitamin D were reduced by 86%, 58%, and 50%, respectively. A selective EP₄ receptor antagonist (EP₄RA) further inhibited TRAP+ MNC formation in both EP₂ +/+ and EP₂ −/− cultures. In cocultures of spleen and calvarial osteoblastic cells, the response to PGE₂ or PTH was reduced by 92% or 85% when both osteoblastic cells and spleen cells were from EP₂− /− mice, by 88% or 68% when only osteoblastic cells were from EP₂ −/− mice and by 58% or 35% when only spleen cells were from EP₂ −/− mice. PGE₂ increased receptor activator of nuclear factor (NF)-kB ligand (RANKL) messenger RNA expression in osteoblastic and bone marrow cell cultures from EP₂ +/+ mice 2-fold but had little effect on cells from EP₂ −/− mice. Spleen cells cultured with RANKL and macrophage colony stimulating factor produced TRAP+ MNC. PGE₂ increased the number of TRAP+ MNC in spleen cell cultures from EP₂ +/+ mice but not in cultures from EP₂ −/− mice. EP₄RA had no effect on the PGE₂ response in spleen cell cultures. PGE₂ decreased the expression of messenger RNA for granulocyte-macrophage colony stimulating factor in spleen cell cultures from EP₂+ /+ mice but had little effect on cells from EP₂ −/− mice. These data demonstrate that the prostaglandin EP₂ receptor plays a role in the formation of osteoclast-like cells in vitro. A major defect in EP₂ −/− mice appears to be in the capacity of osteoblastic cells to stimulate osteoclast formation. In addition, there appears to be a defect in the response of cells of the osteoclastic lineage to PGE₂ in EP₂ −/− mice.