Bicarbonate transport and metabolism are key elements of normal cellular function. Two alternate transcripts of anion exchanger 3 (AE3), full-length (AE3fl) and cardiac (AE3c), are expressed in central nervous system (CNS), where AE3 catalyzes electroneutral Cl(-)/HCO(3)(-) exchange across the plasma membrane of neuronal and glial cells of CNS. Anion exchanger isoforms, AE3fl and AE3c, associate with the carbonic anhydrases (CA) CAII and CAIV, forming a HCO(3)(-) transport metabolon, to maximize HCO(3)(-) flux across the plasma membrane. CAXIV, with catalytic domain anchored to the extracellular surface, is also expressed in CNS. Here physical association of AE3 and CAXIV was examined by coimmunoprecipitation experiments, using mouse brain and retinal lysates. CAXIV immunoprecipitated with anti-AE3 antibody, and both AE3 isoforms were immunoprecipitated using anti-CAXIV antibody, indicating CAXIV and AE3 interaction in the CNS. Confocal images revealed colocalization of CAXIV and AE3 in Müller and horizontal cells, in the mouse retina. Cl(-)/HCO(3)(-) exchange activity of AE3fl was investigated in transiently transfected human embryonic kidney 293 cells, using intracellular fluorescence measurements of BCECF, to monitor intracellular pH. CAXIV increased the rate of AE3fl-mediated HCO(3)(-) transport by up to 120%, which was suppressed by the CA inhibitor acetazolamide. Association of AE3 and CAXIV may represent a mechanism to enhance disposal of waste CO(2) and to balance pH in excitable tissues.