In our present study, we examined the effect of Cl− on rabbit renal brush-border membrane (BBM) phosphate (Pi) uptake. It was found that the Na+-dependent BBM32P 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 BBM36Cl−uptake but did not affect BBM 32P uptake. BBM22Na+uptake was lowered by Cl−replacement in the uptake solution but not by Cl− transport inhibitors. Changes in transmembrane electrical potential altered BBM36Cl−and 32P uptake in directions consistent with a net inward movement of negative and positive charges, respectively. However, the Cl−-dependent BBM Pi uptake was not affected by changes in transmembrane electrical potential. Finally, a similar Cl− dependency of Pi uptake was also found with BBM derived from rat and mouse kidneys. In summary, our study showed that a component of Na+-dependent Pi uptake was also Cl− dependent in rabbit, rat, and mouse renal BBM. The mechanism underlying this Cl− dependency remains to be identified.