The receptor-ligand interaction between the cardiac glycoside Ouabain and purified, membrane-bound as well as micellar Na,K-ATPase is investigated. Calorimetric titrations are carried out with micromolar concentrations of the phosphorylated protein in the presence of Mg2+. The measured heat changes provide evidence for an exothermic, high affinity and specific receptor binding process as well as for a low affinity, nonspecific binding to the lipid part of the nanoparticulate membrane fragments. The degree of lipid binding markedly depends on the lipid composition of the tissue. The measured time course of the heat chan-e resulting from specific binding to the receptor site is unusually slow and is limited by the binding kinetics of the ligand. A course estimation of the Ouabain binding kinetics leads to a rate constant around 10(4) mol(-1) 1 s(-1). Receptor binding is characterized by affinities ranging between 10(7) and 10(8) mol(-1) 1, DeltaH values around -95 kJ mol(-1) and DeltaS values of about - 130 J K-1 mol(-1) at 25 degreesC. The enthalpic contribution is assumed to be mainly due to hydrogen bond formations between the ligand and the receptor site whereas the large, negative entropy change may be attributed to an increased interaction between water and the protein as a consequence of a conformational transition. The evaluation of the titrations provides stoichiometric coefficients around 0.55, which implies that only about 50-60% of the Na,K-ATPase protomers are capable to bind the cardiotonic steroid. This result is consistent with radioactive phosphorylation studies and appears to be a typical feature of kidney-type Na,K-ATPase preparations. Possible implications of this finding are discussed. As a general result, this study demonstrates how simple and suitable calorimetric titrations with micromolar protein concentrations can be for the purpose of a quantitative characterization of a receptor in nanoparticulate membrane systems.