Osteoclasts possess inorganic phosphate (Pi) transport systems to take up external Pi during bone resorption. In the present study, we characterized Pi transport in mouse osteoclast-like cells that were obtained by differentiation of macrophage RAW264.7 cells with receptor activator of NF-kappaB ligand (RANKL). In undifferentiated RAW264.7 cells, Pi transport into the cells was Na+ dependent, but after treatment with RANKL, Na+-independent Pi transport was significantly increased. In addition, compared with neutral pH, the activity of the Na+-independent Pi transport system in the osteoclast-like cells was markedly enhanced at pH 5.5. The Na+-independent system consisted of two components with Km of 0.35 mM and 7.5 mM. The inhibitors of Pi transport, phosphonoformic acid, and arsenate substantially decreased Pi transport. The proton ionophores nigericin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone as well as a K+ ionophore, valinomycin, significantly suppressed Pi transport activity. Analysis of BCECF fluorescence indicated that Pi transport in osteoclast-like cells is coupled to a proton transport system. In addition, elevation of extracellular K+ ion stimulated Pi transport, suggesting that membrane voltage is involved in the regulation of Pi transport activity. Finally, bone particles significantly increased Na+-independent Pi transport activity in osteoclast-like cells. Thus, osteoclast-like cells have a Pi transport system with characteristics that are different from those of other Na+-dependent Pi transporters. We conclude that stimulation of Pi transport at acidic pH is necessary for bone resorption or for production of the large amounts of energy necessary for acidification of the extracellular environment.