In our present study, we examined the effect of Cl on rabbit renal brush-border membrane (BBM) phosphate (P_i) uptake. It was found that the Na⁺-dependent BBM ³²P uptake was significantly inhibited by Cl replacement in the uptake solution with other anions, or by Cl transport inhibitors, including DIDS, SITS, diphenylamine-2-carboxylate (DPC), niflumic acid (NF), and 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB). Intravesicular formate or Cl increased BBM ³⁶Cl uptake but did not affect BBM ³²P uptake. BBM ²²Na⁺ uptake was lowered by Cl replacement in the uptake solution but not by Cl transport inhibitors. Changes in transmembrane electrical potential altered BBM ³⁶Cl and ³²P uptake in directions consistent with a net inward movement of negative and positive charges, respectively. However, the Cl-dependent BBM P_i uptake was not affected by changes in transmembrane electrical potential. Finally, a similar Cl dependency of P_i uptake was also found with BBM derived from rat and mouse kidneys. In summary, our study showed that a component of Na⁺-dependent P_i uptake was also Cl dependent in rabbit, rat, and mouse renal BBM. The mechanism underlying this Cl dependency remains to be identified.