The proton-linked monocarboxylate transporters MCT1-4 play a crucial role in the shuttling of lactate between glycolytic and oxidative cell types in different tissues like muscle and brain. Using heterologous protein expression in Xenopus oocytes, we have recently reported that carbonic anhydrase isoform II (CAII), a ubiquitous enzyme catalyzing the equilibration of carbon dioxide, protons and bicarbonate enhances transport activity of MCT1 and MCT4, while leaving transport activity of MCT2 unaffected. This interaction is independent of CAII catalytic activity, but is mediated by an intramolecular H+-shuttle within the enzyme (1-4). In a further study we could demonstrate that endogenous CAII co-localizes with MCT1 in mouse astrocytes in which it enhances lactate flux by a non-catalytic mechanism (5). Comparison of the protein sequence of MCT1, 2 and 4 revealed potential binding sites for CAII in the C-terminal of MCT1 and 4, but not in MCT2. For identification of the binding sites, truncation and single site mutations were placed in the C-terminal tail of MCT1 and MCT4. The transporters were heterologously expressed in Xenopus oocytes with and without CAII and transport activity was determined with pH-sensitive microelectrodes. Oocytes were surgically removed under sterile conditions from Xenopus laevis females anaesthetized with 1 g/l of 3-amino-benzoic acid ethylester (MS-222) in their bath. The experiments revealed that the acidic amino acid clusters E489EE in MCT1 and E431EE in MCT4 are crucial for the functional interaction with CAII. Direct binding of CAII to MCT1 and MCT4, respectively, via these acidic clusters was confirmed by protein biochemical analysis. To confirm that the inability of CAII to enhance transport activity of MCT2 is due to the absence of an appropriate binding site within the transporters C-terminal, we exchanged the last seven amino acids of the MCT2 C-terminal with the corresponding amino acids of the MCT1 C-terminal (R483DKESSI to P483AEEESP), creating a putative CAII binding site. Expression of the mutant in Xenopus oocytes showed that the insertion of this binding site indeed enabled CAII to enhance transport activity of MCT2. Our results suggest that the modulation of MCT1/4 transport activity by CAII requires direct interaction between transporter and enzyme, which is mediated by a glutamic acid cluster within the C-terminal tail of the MCTs.