The class C L-type calcium (Ca2+) channels have been implicated in many important physiological processes. sere, we have identified a mouse vacuolar H+-ATPase (V-ATPase) G2 subunit protein that bound to the C-terminal domain of the pore-forming alpha (1C) subunit using a yeast two-hybrid screen. Protein-protein interaction between the V-ATPase G subunit and the alpha (1C) subunit was confirmed using in vitro GST pull-down assays and coimmunoprecipitation from intact cells, Moreover, treatment of cells expressing L-type Ca2+ channels with a specific inhibitor of the V-ATPase blocked proper targeting of the channels to the plasma membrane.