To elucidate the mechanism responsible for the establishment of steady state pH at zero net flux (pH infinity) in proximal convoluted tubules, luminal pH was recorded continuously with antimony microelectrodes under three experimental conditions. First: luminal pH in stationary droplets was allowed to reach pH infinity (6.76 +/- 0.07) and then carbonic anhydrase inhibitor benzolamide (3 x 10(-3) mol/l) was superfused on the kidney surface. Following application of benzolamide, luminal pH decreased within seconds (delta pH = -0.27 +/- 0.03 SEM). Second: tubule segments were perfused continuously with MES-buffer containing solution set to a pH of 6.1. Some 1--2 mm distal to the perfusion pipette luminal pH was recorded and was 6.5 +/- 0.04. After superfusion of benzolamide (3 x 10(-3) mol/l) pH decreased (delta pH = -0.15 +/- 0.03). Third: pH in stationary droplets was again allowed to reach PH infinity (6.69 +/- 0.01) and bicarbonate and CO2- free solution (5 mmol/l phosphate set to a pH of 7.4) was microinfused into the adjacent peritubular capillary. Luminal pH again decreased almost immediately (delta pH = -0.23 +/- 0.02). The data are interpreted as evidence for a bicarbonate leak. In a fourth series of experiments, segments of proximal tubules were perfused under benzolamide (0.4 x 10(-6) mol/min) with solutions initially free of bicarbonate or other buffers. In the collected fluid, bicarbonate was determined by a micro-Astrup method. A significant increase of luminal bicarbonate concentration (r = 0.88) indicates a permeability of 0.98 +/- 0.14 x 10(-6) cm2/s of the tubular wall for bicarbonate. Since bicarbonate eventually increases more than 3-fold the equilibrium concentration, collected bicarbonate could not have been formed by H2CO3 or CO2. Bicarbonate enters the luminal fluid and reacts with secreted hydrogen ions to forms carbonic acid. It, therefore, buffers secreted hydrogen ions and increases luminal pH at or below steady state. Inhibition of carbonic anhydrase and lowering of peritubular bicarbonate thus lower pH infinity.