We examined the effect on osteoclast formation of disrupting the prostaglandin G/H synthase genes PGHS-1 and-2. Prostaglandin E₂ (PGE₂) production was significantly reduced in marrow cultures from mice lacking PGHS-2 (PGHS-2^–/–) compared with wild-type (PGHS-2^+/+) cultures. Osteoclast formation, whether stimulated by 1,25-dihydroxyvitamin D₃ (1,25-D) or by parathyroid hormone (PTH), was reduced by 60–70% in PGHS-2^–/– cultures relative to wild-type cultures, an effect that could be reversed by providing exogenous PGE₂. Cultures from heterozygous mice showed an intermediate response. PGHS inhibitors caused a similar drop in osteoclast formation in wild-type cultures. Co-culture experiments showed that supporting osteoblasts, rather than osteoclast precursors, accounted for the blunted response to 1,25-D and PTH. This lack of response appeared to result from reduced expression of RANK ligand (RANKL) in osteoblasts. We cultured spleen cells with exogenous RANKL and found that osteoclast formation was 50% lower in PGHS-2^–/– than in wild-type cultures, apparently because the former cells expressed high levels of GM-CSF. Injection of PTH above the calvaria caused hypercalcemia in wild-type but not PGHS-2^–/– mice. Histological examination of bone from 5-week-old PGHS-2^–/– mice revealed no abnormalities. Mice lacking PGHS-1 were similar to wild-type mice in all of these parameters. These data suggest that PGHS-2 is not necessary for wild-type bone development but plays a critical role in bone resorption stimulated by 1,25-D and PTH.