Monocarboxylate transporter (MCT) isoforms 1–4 are responsible for the electroneutral H⁺-linked transport of high-energy metabolites like lactate and pyruvate. Carbonic anhydrase (CA) is an ubiquitous enzyme catalysing the equilibration of CO₂, H⁺ and HCO₃^-. We have recently shown that transport activity of MCT1 and 4, heterologously expressed in Xenopus oocytes, is supported by cytosolic CAII in a non-catalytic manner (Becker & Deitmer, 2008, J Biol Chem 283, 21655-67; Becker, Klier & Deitmer, 2010, J Membr Biol 234, 125-35). Expressed in Xenopus oocytes, we have now investigated possible interactions of the high-affinity MCT isoform 2 with cytosolic CAII as well as with extracellular CAIV, anchored to the membrane by a glycosyl phosphatidylinositol (GPI) linkage. We could confirm that MCT2 needs embigin as ancillary protein for proper expression and catalytic function. In contrast to MCT1 and 4, activity of MCT2 was not affected by cytosolic CAII. However, extracellular CAIV-WT increased MCT2 transport activity specifically when embigin was coexpressed. The interaction between MCT2 and CAIV was persistent in the nominal absence of CO₂/HCO₃^- as well as in the presence of the CA inhibitor 6-ethoxy-2-benzothiazolesulfonamide (EZA), indicating a non-catalytic facilitation of transport activity by CAIV. Furthermore, two CAIV mutants, the catalytically inactive mutant CAIV-V165Y, and the newly generated CAIV-H88A in which a histidine, serving as intramolecular H⁺-shuttle, was exchanged, increased MCT2 transport activity by about 90% and 75%, respectively, as compared to the enhancement induced by CAIV-WT. Supported by the DFG (GRK 845)