Osteoclasts are the primary cells responsible for bone resorption. They are exposed to high ambient concentrations of inorganic phosphate (Pi) during the process of bone resorption and they possess specific Pi-transport system(s) capable of taking up Pi released by bone resorption. By immunochemical studies and PCR, we confirmed previous studies suggesting the presence of an Na-dependent Pi transporter related to the renal tubular "NaPi" proteins in the osteoclast. Using polyclonal antibodies to NaPi-2 (the rat variant), an approximately 95-kD protein was detected, localized in discrete vesicles in unpolarized osteoclasts cultured on glass coverslips. However, in polarized osteoclasts cultured on bone, immunofluorescence studies demonstrated the protein to be localized exclusively on the basolateral membrane, where it colocalizes with an Na-H exchanger but opposite to localization of the vacuolar H-ATPase. An inhibitor of phosphatidylinositol 3-kinase, wortmannin, and an inhibitor of actin cytoskeletal organization, cytochalasin D, blocked the bone-stimulated increase in Pi uptake. Phosphonoformic acid (PFA), an inhibitor of the renal NaPi-cotransporter, reduced NaPi uptake in the osteoclast. PFA also elicited a dose-dependent inhibition of bone resorption. PFA limited ATP production in osteoclasts attached to bone particles. Our results suggest that Pi transport in the osteoclast is a process critical to the resorption of bone through provision of necessary energy substrates.