The monocarboxylate transporters MCT1 and 4, which are highly expressed in astrocytes, have been suggested to be responsible for the export of lactate to provide neurons with the energy substrate. In the present study, we tested whether carbonic anhydrase II (CAII) can facilitate lactate transport in astrocytes, as previously shown in Xenopus oocytes (Becker & Deitmer, 2008). In CAII knockout mice a significant reduction in the rate of lactate-induced acidification was found in astrocytes of acute cerebellar slices. Blocking CA catalytic activity had no effect on H⁺ flux, suggesting a non-catalytic facilitation of transport activity by CAII. The data could be confirmed by uptake experiments in mouse astrocytes culture: Knock-down of CAII and CAIV by siRNA led to a significant decrease in lactate flux, while inhibition of CA catalytic activity had no effect. In situ proximity ligation assays indicated close proximity (<40 nm) of MCT1 and CAII as intrinsically expressed in cultured astrocytes. Single site mutations of MCT1, heterologously expressed in Xenopus oocytes, revealed that the interaction between MCT1 and CAII seems to be facilitated by a cluster of three glutamate residues in the C-terminal of MCT1. While catalytic activity of CAII is apparently not necessary for enhancement of MCT1 transport activity, removal of the intramolecular H⁺-shuttle, His64, in CAII abolished the interaction between the two proteins. Our results suggest that CAII, directly bound to MCT1, can facilitate lactate flux in astrocytes by acting as a "proton collecting antenna" which would facilitate proton movement at the pore of the transporter.