Prostaglandin E2 (PGE2) and the cytokines interleukin (IL) 1alpha and tumor necrosis factor (TNF)alpha increase bone resorption in vivo, but the effect of these agents on osteoclastic bone resorption has never been studied in an in vitro human system. Our recently described human bone marrow culture system, in which osteoclasts are generated (vitronectin and calcitonin receptor-positive cells which resorb bone), was used to study the effects of these agents. Addition of indomethacin to macrophage colony-stimulating factor (M-CSF)-treated cultures nearly abolished osteoclast parameters, indicating that prostaglandins are virtually essential for human osteoclast formation. Additionally, PGE2 dose responsively increased osteoclast numbers and bone resorption. The effects of M-CSF and PGE2 are independent, as demonstrated by unaltered PGE2 concentrations in culture supernatants in spite of the dose-responsive increase in osteoclast parameters in response to M-CSF. The generation of osteoclasts in the presence of PGE2 occurred in favor of CD 14-positive macrophage formation. IL 1alpha and TNFalpha increased osteoclast parameters in a dose-responsive manner. Maximum stimulation yielded culture supernatant levels of PGE2 approximately the same as those concentrations of exogenous PGE2 that dramatically induced osteoclast formation. This osteoclast-inducing effect was inhibited both by indomethacin and by the specific inhibitor of inducible prostaglandin G/H synthase, NS398, and this was reversed by addition of exogenous PGE2. These results demonstrate unequivocally that IL 1alpha and TNFalpha enhance human osteoclast formation and suggest that they mediate their effects through PGE2.