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 cell with receptor activator of nuclear factor kappa B ligand (RANKL). In undifferentiated RAW264.7 cells, Pi transport into the cells was sodium-dependent, but, after treatment of RANKL, the sodium-independent Pi transport system was significantly increased. In addition, compared to neutral pH, the activity of the sodium-independent Pi transport system in the osteoclast-like cells was markedly enhanced at pH 5.5. The sodium-independent system consisted of two components with K_m's of 0.35 mM and 7.5 mM. The inhibitors of Pi transport, phosphonoformic acid and arsenate substantially decreased Pi transport. Inhibitors of anion transport, including 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid and 4,4'-dinitrostilbene-2,2'-disulufonic acid disodium salt, had no effect on Pi transport. In contrast, proton ionophores, nigericin and calbonylcyanide p-trifluorome thxyphenylhydrazone, and a potassium ionophore, valinomycin, significantly suppressed the Pi transport activity. Analysis of BCECF fluorescence indicated that Pi transport in the osteoclast-like cells is coupled to a proton transport system. In addition, elevation of extracellular potassium ion stimulated Pi transport, suggesting that the membrane voltage is involved in regulation of Pi transport activity. We did not detect the expression of sodium-dependent Pi transporter (types I, IIb and IIc) transcripts, but those of the type IIa and type III transporter were observed. Finally, bone particles significantly increased sodium-independent Pi transport activity in the osteoclast-like cells. Thus, the osteoclast-like cells have a Pi transport system with characteristics that are different than other sodium-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.