Chronic metabolic acidosis stimulates net calcium efflux from bone due to increased osteoclastic bone resorption and decreased osteoblastic collagen synthesis. Previously we have determined that incubation of neonatal mouse calvariae in medium simulating physiologic metabolic acidosis leads to a significant, cyclooxygenase-dependent, increase in RNA for bone cell receptor activator of NFB ligand (RANKL) as compared to incubation in neutral pH medium. In this study we tested the hypothesis that the acid-mediated increase in RANKL expression is a primary mechanism for the stimulated osteoclastic resorption. Acid medium increased the medium concentration of sRANKL without altering the concentration of the decoy receptor osteoprotegerin (OPG). Inhibition of the RANKL pathway with concentrations of OPG up to 25 ng/ml, far greater than physiologic, did not significantly decrease the robust acid-induced Ca efflux from bone, nor did incubation of the calvariae with a different inhibitor, RANK/Fc (up to 50 ng/ml). Thus acid-induced net Ca efflux appears to involve mechanisms in addition to the RANK/RANKL pathway. Osteoblasts also produce tumor necrosis factor - (TNF-), another cytokine that stimulates the maturation and activity of osteoclasts. Incubation of calvariae in acid medium caused a significant increase in TNF- levels. Incubation of calvariae with anti-TNF (up to 250 ng/ml) did not significantly decrease acid-induced net Ca efflux. However, the combination of RANK/Fc plus anti-TNF caused a significant but sub-total reduction in acid-induced Ca efflux, while the combination of RANK/Fc plus an isotype-matched control for the anti-TNF had no effect on Ca release. Thus simultaneous inhibition of RANKL and TNF- is necessary to reduce acid-induced, cell-mediated net Ca efflux from bone; however, additional osteoblast-produced factors must also be involved in acid-induced, cell-mediated bone resorption.