Anion exchanger 1 (AE1) is the plasma membrane Cl−/HCO3− exchanger of erythrocytes. Carbonic anhydrases (CA) provide substrate for AE1 by catalyzing the reaction, H2O + CO2 ↔ HCO3− + H+. The physical complex of CAII with AE1 has been proposed to maximize anion exchange activity. To examine the effect of CAII catalysis on AE1 transport rate, we fused either CAII-wild type or catalytically inactive CAII-V143Y to the cytoplasmic COOH terminus of AE1 to form AE1.CAII and AE1.CAII-V143Y, respectively. When expressed in transfected human embryonic kidney 293 cells, AE1.CAII had a similar Cl−/HCO3− exchange activity to AE1 alone, as assessed by the flux of H+ equivalents (87 ± 4% vs. AE1) or rate of change of intracellular Cl− concentration (93 ± 4% vs. AE1), suggesting that CAII does not activate AE1. In contrast, AE1.CAII-V143Y displayed transport rates for H+ equivalents and Cl− of 55 ± 2% and of 40 ± 2%, versus AE1. Fusion of CAII to AE1 therefore reduces anion transport activity, but this reduction is compensated for during Cl−/HCO3− exchange by the presence of catalytically active CAII. Overexpression of free CAII-V143Y acts in a dominant negative manner to reduce AE1-mediated HCO3− transport by displacement of endogenous CAII-wild type from its binding site on AE1. To examine whether AE1.CAII bound endogenous CAII, we coexpressed CAII-V143Y along with AE1 or AE1.CAII. The bicarbonate transport activity of AE1 was inhibited by CAII-V143Y, whereas the activity of AE1.CAII was unaffected by CAII-V143Y, suggesting impaired transport activity upon displacement of functional CAII from AE1 but not AE1.CAII. Taken together, these data suggest that association of functional CAII with AE1 increases Cl−/HCO3− exchange activity, consistent with the HCO3− transport metabolon model.