The mammalian kidney isoform of the essential chloride-bicarbonate exchanger AE1 differs from its erythrocyte counterpart, being shorter at its N terminus. It has previously been reported that the glycolytic enzyme GAPDH interacts only with erythrocyte AE1, by binding to the portion not found in the kidney isoform. (Chu H, Low PS. Biochem J 400:143-151, 2006). We have identified GAPDH as a candidate binding partner for the C terminus of both AE1 and AE2. We show that full-length AE1 and GAPDH coimmunoprecipitated from both human and rat kidney as well as from Madin-Darby canine kidney (MDCK) cells stably expressing kidney AE1, while in human liver, AE2 coprecipitated with GAPDH. ELISA and glutathione S-transferase (GST) pull-down assays using GST-tagged C-terminal AE1 fusion protein confirmed that the interaction is direct; fluorescence titration revealed saturable binding kinetics with Kd 2.3±0.2 μM. Further GST precipitation assays demonstrated that the D902EY residues in the D902EYDE motif located within the C terminus of AE1 are important for GAPDH binding. In vitro GAPDH activity was unaffected by C-terminal AE1 binding, unlike in erythrocytes. Also, differently from red cell N-terminal binding, GAPDH-AE1 C-terminal binding was not disrupted by phosphorylation of AE1 in kidney AE1-expressing MDCK cells. Importantly, small interfering RNA knockdown of GAPDH in these cells resulted in significant intracellular retention of AE1, with a concomitant reduction in AE1 at the cell membrane. These results indicate differences between kidney and erythrocyte AE1/GAPDH behavior and show that in the kidney, GAPDH is required for kidney AE1 to achieve stable basolateral residency.