Functionally active Na+,K+-ATPase isozymes containing three types of the catalytic subunits (α1, α2, and α3) were obtained from calf brain by two methods: selective removal of contaminating proteins according to Jorgensen (1974) and selective solubilization of the enzyme with subsequent reformation of the membrane structure according to Esmann (1988). All preparations were characterized with respect to ouabain-inhibition constants. The presence of the cytoskeleton protein tubulin (β3 isoform) in the high-molecular-weight complex of Na+,K+-ATPase α3β1 isozyme from brain stem axolemma and the junction between Na+,K+-ATPase α3 subunit and tubulin β3 subunit are shown for the first time.