Multiple forms of the catalytic subunit of the (Na+,K+)ATPase have been identified in rat brain. While two of them (.alpha.1 and .alpha.2) have been well characterized, the third form (.alpha.3) of these catalytic subunits only recently has been described by cDNA cloning; the corresponding polypeptide has not been isolated. In this paper it is shown that rat brain contains the .alpha.3 chain. The catalytic subunits of the (Na+,K+)ATPase from rat brain axolemma were purified by SDS-PAGE and subjected to formic acid cleavage. Amino acid sequence analysis of the resulting fragments revealed that axolemma has the .alpha.3 form of the catalytic subunit. In addition, .alpha.3-specific antiserum was raised in rabbits immunized with a synthetic peptide. Immunoblotting with this antiserum revealed that the .alpha.3 form of the (Na+,K+)ATPase is present also in whole brain microsomes. In SDS-PAGE, the mobilities of the three catalytic subunits of brain (Na+,K+)ATPase follow the order .alpha.1 > .alpha.2 > .alpha.3. Determination of the ouabain-inhibitable ATPase activity indicates that if the .alpha.3 form of the (Na+,K+)ATPase is able to hydrolyze ATP, it is present in a form of the enzyme with a high affinity for this cardiac glycoside and is similar to the .alpha.2 form in this respect.