Although hypercholesterolemia is a strong risk factor for cardiovascular disease it has in some instances paradoxically been associated with reduced infarct size and preserved contractile function in isolated hearts after ischemia and reperfusion. To elucidate potential cellular protective mechanisms, myocytes of hypercholesterolemic apolipoprotein E -deficient (ApoE-/-) and wild-type mice were subjected to hypoxic metabolic inhibition (I) with subsequent reoxygenation (R). Intracellular Ca²⁺ concentration ([Ca²⁺]_i) and pH [pH_i] were monitored as well as cell length and arrhythmic events. Force measurements in papillary muscles were also recorded and myocardial expression of Na⁺/H⁺ exchanger 1 (NHE1) and three Ca²⁺ handling proteins (sarcoendoplasmic Ca²⁺-ATPase, Na⁺/Ca²⁺ exchanger, plasma membrane Ca²⁺-ATPase) was quantified. After 30 min of I and 35 min of R, Ca²⁺ overload was more pronounced in wild-type cells (P<0.05). In these myocytes pH_i also dropped faster and remained below those values determined in ApoE-/- cells (P<0.05). Furthermore, more wild-type myocytes remained in a contracted state (P<0.05). This group also showed a higher incidence of arrhythmic events during R (P<0.05). No group difference was found in the expression of the Ca²⁺ handling proteins. However, NHE1 protein was down-regulated in hearts of ApoE-/- mice (P<0.05). Histologic results depict hyperplasia in ApoE-/- hearts without atherosclerosis of the coronaries. Contractile dysfunction was not observed in papillary muscles from ApoE-/- hearts. Our results suggest that down-regulated myocardial NHE1 expression in hypercholesterolemic ApoE-/- mice could have contributed to increased tolerance to I/R. It remains to be elucidated whether NHE1 down-regulation is a unique feature of these genetically altered animals.