The thermal unfolding and domain structure of Na⁺/K⁺‐ATPase from pig kidney were studied by high‐sensitivity differential scanning calorimetry (HS‐DSC). The excess heat capacity function of Na⁺/K⁺‐ATPase displays the unfolding of three cooperative domains with midpoint transition temperatures (T_d) of 320.6, 327.5, 331.5 K, respectively. The domain with T_d = 327.5 K was identified as corresponding to the β subunit, while two other domains belong to the α subunit. The thermal unfolding of the low‐temperature domain leads to large changes in the amplitude of the short‐circuit current, but has no effect on the ATP hydrolysing activity. Furthermore, dithiothreitol or 2‐mercaptoethanol treatment causes destruction of this domain, accompanied by significant disruption of the ion transporting function and a 25% loss of ATPase activity. The observed total unfolding enthalpy of the protein is rather low (≈ 12 J·g⁻¹), suggesting that thermal denaturation of Na⁺/K⁺‐ATPase does not lead to complete unfolding of the entire molecule. Presumably, transmembrane segments retain most of their secondary structure upon thermal denaturation. The binding of physiological ligands results in a pronounced increase in the conformational stability of both enzyme subunits.