Cisplatin treatment increases the excretion of inorganic phosphate in vivo. However, the mechanism by which cisplatin reduces phosphate uptake through renal proximal tubular cells has not yet been elucidated. We examined the effect of cisplatin on Na⁢-dependent phosphate uptake in opossum kidney (OK) cells, an established proximal tubular cell line. Cells were exposed to cisplatin for an appropriate time period and phosphate uptake was measured using [32P]-phosphate. Changes in the number of phosphate transporter in membranes were evaluated by kinetic analysis, [14C]phosphonoformic acid binding, and Western blot analysis. Cisplatin inhibited phosphate uptake in a time- and dose-dependent manner, and also the Na⁢-dependent uptake without altering Na⁢-independent uptake. The cisplatin inhibition was not affected by the hydrogen peroxide scavenger catalase, but completely prevented by the hydroxyl radical scavenger dimethylthiourea. Antioxidants were ineffective in preventing the cisplatin-induced inhibition of phosphate uptake. Kinetic analysis indicated that cisplatin decreased Vmax of Na⁢-dependent phosphate uptake without any change in the Km value. Na⁢-dependent phosphonoformic acid binding was decreased by cisplatin treatment. Western blot analysis showed that cisplatin caused degradation of Na⁢-dependent phosphate transporter protein. Taken together, these data suggest that cisplatin inhibits phosphate transport in renal proximal tubular cells through the reduction in the number of functional phosphate transport units. Such effects of cisplatin are mediated by production of hydroxyl radicals.