The plasma membrane Ca2+-ATPase is a well known enzyme in eucaryotes able to extrude calcium to the extracellular space in order to restore intracellular calcium to very low levels. This ATPase needs plasma membrane lipids such as acidic phospholipids in order to maintain its activity. In this study, we investigated the role that calcium and cholesterol play on the thermal stability of the Ca2+-ATPase isolated from cardiac sarcolemma and erythrocyte membranes. Calcium showed a stabilizing and protective effect when the enzyme was exposed to high temperatures. This stabilizing effect showed by calcium was potentiated in the presence of cholesterol. These protection effects were reflected on several thermodynamic parameters such as T50, ▵Hvh and apparent ▵G, indicating that calcium might induce a conformational change stabilized in the presence of cholesterol that confers enzyme thermostability. The effect shown by cholesterol on ▵Hvh and apparent ▵H‡ open the possibility that this lipid decreases cooperativity during the induced transition. Despite that a binding site for cholesterol has not been identified in the plasma membrane Ca2+-ATPase, our results supports the proposal that this lipid interacts with the enzyme in a direct fash