Functionally active preparations of Na+,K+-ATPase isozymes from calf brain that contain catalytic subunits of three types (α1, α2, and α3) were obtained using two approaches: a selective removal of contaminating proteins by the Jorgensen method and a selective solubilization of the enzyme with subsequent reconstitution of their membrane structure by the Esmann method. The ouabain inhibition constants were determined for the isozymes. The real isozyme composition of the Na+pump from the grey matter containing glial cells and the brain stem containing neurons was determined. The plasma membranes of glial cells were shown to contain mainly Na+,K+-ATPase of the α1β1 type and minor amounts of isozymes of the α2β2(β1) and the α3β1(β2) type. The axolemma contains α2β1 and α3β1 isozymes. A carbohydrate analysis indicated that α1β1 enzyme preparations from the brain grey matter substantially differ from the renal enzymes of the same composition in the glycosylation of the β1 isoform. An enhanced sensitivity of the α3 catalytic subunit of Na+,K+-ATPase from neurons to endogenous proteolysis was found. A point of specific proteolysis in the amino acid sequence PNDNR492↓ Y493was localized (residue numbering is that of the human α3 subunit). This sequence corresponds to one of the regions of the greatest variability in α1-, α2-, α3-, and α4-subunits, but at the same time, it is characteristic of the α3 isoforms of various species. The presence of the β3 isoform of tubulin (cytoskeletal protein) was found for the first time in the high-molecular-mass Na+,K+-ATPase α3β1 isozyme complex isolated from the axolemma of brain stem neurons, and its binding to the α3 catalytic subunit was shown.