The alpha catalytic subunits of Na+/K(+)-ATPase were isolated from the kidney and brain of rats (alpha 1 and alpha 2, respectively). The antisera raised against these subunits were used as probes to analyze the isoform of catalytic subunits of Na+/K(+)-ATPase in various tissues of rats. Of 27 rat tissues examined, most had a catalytic subunit identical to alpha 1 but some, such as the nervous and muscle tissues, had both alpha 1 and alpha 2 isoforms as judged by their reactivities to antisera and their electrophoretic mobility. We found that the submandibular gland contained a new electrophoretic variant of immunoreactive alpha subunit (designated alpha(S) in this report) in addition to alpha 1 identical to those found in kidney and brain. The new variant, alpha(S), strongly cross-reacted with anti-alpha 1 antiserum, but to a lesser extent with anti-alpha 2 antiserum. The alpha(S) had a molecular mass which was found to be slightly less (approx. 90 kDa) than brain and kidney alpha 1. We examined whether or not the alpha(S) is formed by proteolytic cleavage of alpha subunits during preparation and concluded that this is not the case. The alpha(S) reacted with [gamma-32P]ATP, resulting in the formation of radioactive alpha subunit which was stabilized by 2 mM ouabain but which was labile in the presence of 70 mM potassium chloride. Since N-terminal amino acid sequence of alpha(S) protein [G()DKY()PAAVS] corresponds exactly and uniquely with the sequence of the alpha 1 chain between residues 1 and 11, it is very probable that alpha(S) protein originated from alpha 1 protein following the post-translational processing.