Cholesterol has been shown to regulate the activity of several membrane proteins. Although this phenomenon represents an important factor in the regulation of ion homeostasis, insights are needed to fully understand the role of this lipid in cell function in order to better comprehend the effect of bilayer components upon membrane function. Since evolution has shaped the composition of the membrane bilayer, it becomes of interest to study these changes in parallel with the many functions of membranes such as ion transport. The present study employing a plasma membrane preparation obtained from calf ventricular muscle demonstrates that cholesterol partially inhibits the Ca(2+),Mg(2+)-ATPase as the catalytic function of the calcium pump, when incubation reaction temperatures are below 42 degrees C. In contrast, when incubation reaction temperatures are above 42 degrees C, cholesterol apparently promotes enzyme stabilization reflected in higher activity. Although the activation energy values for the enzyme are almost the same at ranges between 15 and 40 degrees C, the use of elevated temperatures promote higher enzyme inactivation rates in control than in cholesterol enriched membranes. Cholesterol apparently is promoting stabilization that in turn protects the enzyme against thermal inactivation. This protective effect is reflected in a decrease of inactivation rate values and energy released during enzyme catalysis. The modification of many membrane properties throughout million of years made it possible for new evolutionary driving forces to show themselves as new characteristics in eukaryotes such as the one discussed in this study, dealing with the presence of cholesterol in the cell membrane directly associated to the promotion of protein thermostability.