The Ca 2+-ATPase from sarcoplasmic reticulum (SR) membranes couples the Ca 2+ transport to ATP hydrolysis through phosphorylation in its cytoplasmic catalytic domain. Interactions between protein domains and the role of monomer–monomer interactions remain unclear. Here, we report a differential scanning calorimetric study of the thermal unfolding of this protein. In the pH range 6–8, thermal unfolding of the Ca 2+-ATPase in glycogen phosphorylase-free SR membranes shows a major endothermic peak with a critical temperature midpoint ranging between 51 and 55°C, depending on pH, Ca 2+, Mg 2+-ADP and KCl concentrations. The enthalpy change of the overall unfolding process ranged between 250 and 300 kcal/mol of Ca 2+-ATPase monomer. Thermal denaturation of the Ca 2+-ATPase in SR membranes is well fitted to an irreversible process that can be rationalized in terms of a non-two state process, N (native)⇌I (intermediate)→D (denatured). Thermodynamic analysis show that this protein has a compact structure, implying a tight structural interconnection between catalytic and Ca 2+ transport domains. The apparent cooperative unit, defined by the van ’t Hoff enthalpy to the overall unfolding enthalpy ratio, increased from 1.1 at pH 6 to 1.8 at pH 8, showing that monomer–monomer interactions are stronger at weakly basic pH than at weakly acidic pH. While micromolar Ca 2+ concentrations had only a weak effect on the cooperativity of the unfolding process, this is clearly increased by millimolar Mg 2+-ADP. In addition, high ionic strength lowered the apparent cooperative unit to approximately 1.0 in the pH range 6–8. Taken together, these results suggest that protein–protein interactions are altered by variables that modulate the catalytic activity of this enzyme.