The expression of the canine alpha 2 and 3 subunit isoenzymes of NA,K-ATPase has been investigated in plasma membranes isolated from dog heart, brain and kidney by immunoblotting, employing polyclonal anti rat fusion protein, and enzymological techniques. Western blot analysis revealed with purified dog membrane Na,K-ATPase preparations, one immunoreactive signal with rat specific alpha 3 antisera in cardiac tissues, and two immunoreactive signals with rat alpha 2 and alpha 3 antisera in cerebral tissues. These findings suggested the specific expression of alpha 3 polypeptide in dog heart (99 kDa), whereas dog brain expressed the alpha 2 and 3 polypeptides. The stained bands were superimposed. The antibody to rat brain alpha 1 fusion protein did not cross-react with dog antigens whatever the three tissues tested. Expression of the alpha 3-subunit isoform in dog heart membranes was consistent with a high affinity digitoxigenin-sensitive class of Na,K-ATPase (IC50 = 7 +/- 2 nM). A single component with low affinity to digitoxigenin (IC50 = 110 +/- 10 nM) characterized the alpha 1 kidney form. The mixture of alpha 2 and alpha 3 isoforms in dog brain exhibited an apparent affinity for digitoxigenin (IC50 = 17 +/- 5 nM) lower than the heart. The sodium dependences of the high affinity digitoxigenin sites were for the cardiac alpha 3 form (K0.5 = 10 +/- 1.9 mM) and for the cerebral alpha 2 and alpha 3 mixture (K0.5 19.6 +/- 4.9 mM). The sensitivities for Na+ of the low affinity sites (alpha 1) were: 6.7 +/- 1.4 mM, 6.3 +/- 1.2 mM and 11.6 +/- 2.9 mM in heart, brain and kidney respectively. This is the first report of the catalytic characteristics of the alpha 3 subunit isoenzyme in canine cardiac plasma membranes.