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 CO2 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 CO2 reabsorption decreased by 80% following acetazolamide, from an average of 805 to 168 pmol/min. In contrast, acetazolamide decreased whole kidney fractional total CO2 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.