1,25-Dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) and prostaglandin E 2 (PGE 2 ) are known to influence osteoclast formation indirectly through their effects on osteoblasts. To determine whether 1,25(OH) 2 D 3 and PGE 2 also have a direct effect on circulating osteoclast precursors, these factors were added to long-term cultures of human peripheral blood mononuclear cells (PBMCs) in the presence of osteoprotegerin ligand and macrophage colony-stimulating factor (M-CSF) (+/-dexamethasone). The number of TRAP + and VNR + multinucleated cells and the area of lacunar resorption were decreased when 1,25(OH) 2 D 3 alone was added. A marked increase in resorption pit formation was noted when the combination of 1,25(OH) 2 D 3 and dexamethasone was added to PBMC cultures. Dose-dependent inhibition of osteoclast formation and lacunar resorption was seen when PGE 2 was added to PBMC cultures in both the presence and the absence of dexamethasone. Thus, 1,25(OH) 2 D 3 and PGE 2 not only influence osteoclast formation in the presence of bone stromal cells but also act directly on circulating osteoclast precursors to influence osteoclast differentiation.