Na,K-ATPase transports Na+ and K+ across cell membranes and consists of α- and β-subunits. Na,K-ATPase also associates with small FXYD proteins that regulate the activity of the pump. We have used cryoelectron microscopy of two-dimensional crystals including data to 8 Å resolution to determine the three-dimensional (3-D) structure of renal Na,K-ATPase containing FXYD2, the γ-subunit. A homology model for the α-subunit was calculated from a Ca2+-ATPase structure and used to locate the additional β- and γ-subunits present in the 3-D map of Na,K-ATPase. Based on the 3-D map, the β-subunit is located close to transmembrane helices M8 and M10 and the γ-subunit is adjacent to helices M2 and M9 of the α-subunit.