The extent to which bicarbonate reabsorption in the rat proximal convoluted tubule depends on carbonic anhydrase has been examined by in vivo microperfusion and the measurement of total CO2 concentration by microcalorimetry. Tubules were perfused with an ultrafiltrate-like solution at 13 nl/min, and volume reabsorptive rate (J(v)) was measured using [14C]inulin. Addition of either 800 or 100 μM acetazolamide to the perfusion solution completely inhibited the reabsorption of total CO2. The control total CO2 reabsorptive rate (J(KCO2)) was 147 ± 23 pmol/mm.min, and acetazolamide reduced J(tCO2) to -3 ± 5 pmol/mm.min. Acetazolamide reduced J(v) by 65% from a control of 2.3 ± 0.4 to 0.8 ± 0.1 nl/mm.min. The dose-response curve for acetazolamide showed that the I50 for inhibition of J(tCO2) was 4 μM. The inactive congener of acetazolamide, t-butyl acetazolamide, did not reduce J(v) or inhibit bicarbonate reabsorption, indicating that the effect of acetazolamide on J(tCO2) was specific for carbonic anhydrase inhibition. Since bicarbonate reabsorption was completely blocked by carbonic anhydrase inhibition, there is no need to postulate either carbonic acid recycling or carbonic anhydrase-independent bicarbonate reabsorption.