Several acid-/base-coupled transporters show an interaction with carbonic anhydrase II (CAII), which leads to an enhancement of transport activity. Carbonic anhydrase regulates the concentration of the substrate of the sodium bicarbonate cotransporter (NBCe1) by catalyzing the hydration of CO₂ and the dehydration of HCO₃^-. We have previously shown an increase in NBCe1 activity after injection and co-expression of CAII in Xenopus oocytes (Becker and Deitmer, 2007, J Biol Chem 282:13508–13521). We have now studied three different CA isoforms, CAI, CAII and CAIII, and several mutants of CAII, expressed in Xenopus oocytes alone or together with NBCe1. All three CA isoforms showed similar catalytic activity as determined by the rate of intracellular proton concentration change, measured with H⁺-selective microelectrodes. All CAs were able to increase the NBCe1 transport activity, as demonstrated by an increased rate of rise in intracellular sodium concentration and membrane current in two-electrode voltage-clamp. In vitro measurements of CA activity by mass spectrometry indicated significant differences between CA isoforms. Two CAII mutants, altered in their intramolecular proton shuttling pathway, CAII-H64A and CAII-Y7F, showed significant catalytic activity and also enhanced NBCe1 transport activity. The effect of CAI, CAII and CAII mutants could be reversed by blocking CA activity with ethoxyzolamide (EZA; 10 mM), while the effect of the less EZA-sensitive CAIII was reduced partially. The catalytically inactive mutant CAII-V143Y showed no effect on NBCe1 transport activity. Our results indicate that the ability of CA to enhance NBCe1 transport activity depends primarily on its catalytic activity. Supported by the DFG (GRK 845)