Formate stimulates sodium chloride and fluid reabsorption in kidney proximal tubule; however, the exact cellular mechanism of this effect remains unknown. We hypothesized that the primary target of formate is the apical Na(+)/H(+) exchanger. Here, we demonstrate that formate directly enhances the apical Na(+)/H(+) exchanger (NHE3) activity in mouse kidney proximal tubule. In the absence of CO(2)/HCO(3)(-), addition of formate (500 microM) to the bath and lumen of microperfused mouse kidney proximal tubule caused significant intracellular alkalinization, with intracellular pH (pH(i)) increasing from baseline levels 7.17 +/- 0.01 to 7.55 +/- 0.01 (P < 0.001, n = 14), with a Delta pH of 0.38 +/- 0.02. Removal of luminal chloride did not block cell pH alkalinization by formate (baseline pH of 7.26 +/- 0.01 to 7.53 +/- 0.01 with formate, P < 0.001, n = 10), indicating that the apical Cl(-)/OH(-) exchanger was not the primary mediator of the effect of formate on cell pH. However, removal of sodium from the lumen or addition of EIPA completely prevented cell pH alkalinization. Addition of formate to the lumen and bath in the outer medullary collecting duct, which does not express any apical Na(+)/H(+) exchanger, did not cause any cell pH alkalinization. At lower concentrations (50 microM), formate caused significant pH(i) alkalinization in proximal tubule cells, with pH(i) increasing from baseline levels 7.15 +/- 0.02 to 7.36 +/- 0.02 (P < 0.02, n = 11). Acetate, at 50 microM, had no effect on pH(i). Formate's effect was observed both in the absence and presence of CO(2)/HCO(3)(-) in the media. We conclude that formate stimulates the apical Na(+)/H(+) exchanger NHE3 in the kidney proximal tubule. We propose that formate stimulation of chloride reabsorption in the proximal tubule is indirect and is secondary to the activation of apical Na(+)/H(+) exchanger NHE3, which then leads to the stimulation of the apical chloride/base exchanger.