The purpose of the present free-flow micropuncture study was to determine the fraction of bicarbonate reabsorption in the superficial proximal convoluted tubule that is mediated by the enzyme carbonic anhydrase by using a potent inhibitor of carbonic anhydrase, acetazolamide. Acetazolamide was measured using high-pressure liquid chromatography in the glomerular ultrafiltrate and end-proximal tubule fluid of the Munich-Wistar rat. The acetazolamide concentration in Bowman's space after 20 mg/kg (prime and per hr) intravenously infused acetazolamide was 0.8 x 10 -4 M and after 50 mg/kg was 1.8 x 10 -4 M. Such concentrations have been shown to maximally inhibit bicarbonate reabsorption in previous microperfusion studies. The acetazolamide concentration rose along the length of the tubule primarily due to secretion. Total CO 2 concentration, as determined by microcalorimetry, and inulin concentration were also measured in Bowman's space and the end-proximal tubule. Acetazolamide 20 or 50 mg/kg decreased absolute proximal water reabsorption by 50% from control values, from 16 to 8 nl/min. Absolute proximal total CO 2 reabsorption decreased by 80% following acetazolamide, from an average of 805 to 168 pmol/min. In contrast, acetazolamide decreased whole kidney fractional total CO 2 reabsorption by only 25%. The authors conclude that 80% of bicarbonate reabsorption in the superficial proximal convoluted tubule of the rat is mediated by carbonic anhydrase and is inhibitable by acetazolamide.