Isolated microperfused $3 segments of rabbit renal proximal tubule were investigated with pH-sensitive double-barrelled intracellular microelectrodes to determine whether the C1-/base exchanger, which we have previously identified in the basolateral cell membrane of this segment requires HCO3-or can also work in CO2/HCO 3-free conditions. Cell pH (pHi) was measured in response to sudden substitution of bath C1-by gluconate. In control solutions containing 25 mmol/1 HCO3 pHi increased initially by 5.0 + 0.3 • 10 .3 unit/s but after perfusion with COJHCO3--free solutions pHi of the same ceils increased only by 1.3 _+ 0.2 • 10 -3 unit/s in response to CI-substitution. From measurements of the cellular buffering power it was calculated that the control base flux had fallen drastically from 3.7 + 0.3 to 0.3 + 0.1 • 10 -12 mol/s.cm tubule length. To test whether the remaining flux might have resulted from metabolic CO2, oxidative metabolism was poisoned with cyanide (5 mmol/1). This abolished the pH change (April) in COz/I-ICO3--free solutions, but did not affect the pH shift in the presence of HCO 3-. The data indicate that basolateral Cl-foase exchange in $3 segment requires HCO3-to operate. A model in which HCO3 absorption proceeds in form of OH-and CO2 can be largely excluded.