The equilibrium CO2+H2O right arrow over left arrow H++HCO3- had made it impossible to determine how isolated changes in basolateral CO2 ([CO2]) or HCO3- concentration ([HCO3-]), at a fixed basolateral pH, modulate renal HCO3- or reabsorption. In the present study, we have begun to address this issue by measuring HCO3- reabsorption (JHCO3) and intracellular pH (pHi) in isolated perfused rabbit S2 proximal tubules exposed to three different basolateral (bath) solutions: 1) equilibrated 5% CO2/22 mM HCO3-/pH 7.40, 2) an out-of-equilibrium (OOE) solution containing 5% CO2/pH 7.40 but minimal HCO3- ("pure CO2"), and 3) an OOE solution containing 22 mM HCO3-/pH 7.40 but minimal CO2 ("pure HCO3-"). Tubule lumens were constantly perfused with equilibrated 5% CO2/22 mM HCO3-. Compared with the equilibrated bath solution (JHCO3 = 76.5 +/- 7.7 pmol.min-1.mm-1, pHi = 7.09 +/- 0.04), the pure CO2 bath solution increased JHCO3 by approximately 25% but decreased pHi by 0.19. In contrast, the pure HCO3- bath solution decreased JHCO3 by 37% but increased pHi by 0.24. Our data are consistent with two competing hypotheses: 1) the isolated removal of basolateral HCO3- (or CO2) causes a pHi decrease (increase) that in turn raises (lowers) JHCO3; and 2) HCO3- removal raises JHCO3 by reducing inhibition of basolateral Na/HCO3 cotransport and/or reducing HCO3- backleak, whereas CO2 removal lowers JHCO3 by reducing stimulation of a CO2 sensor.