While several transgenic mouse models exhibit improved contractile characteristics in the heart, less is known about how these changes influence energy metabolism, specifically the balance between carbohydrate and fatty acid oxidation. In the present study we examine glucose and fatty acid oxidation in transgenic mice generated to overexpress SERCA which have an enhanced contractile phenotype. Energy substrate metabolism was measured in isolated working hearts using radiolabelled glucose and palmitate. We also examined oxygen consumption to see if SERCA overexpression is associated with increased oxygen utilization. Since SERCA is important in calcium handling within the cardiac myocyte we examined cytosolic calcium transients in isolated myocytes using indo-1 and mitochondrial calcium levels using Pericam, an adenovirally-expressed mitochondrially-targeted ratiometric calcium indicator. Oxygen consumption did not differ between WT and SERCA groups; however, we were able to show an increased utilization of glucose for oxidative metabolism and a corresponding decreased utilization of fatty acids in the SERCA group. Cytosolic calcium transients were increased in myocytes isolated from SERCA mice and they show a faster rate of decay of the calcium transient. Accompanying these observations we noted increased levels of mitochondrial calcium in the SERCA group which was associated with an increase in the active form of the pyruvate dehydrogenase complex. As an increase in mitochondrial calcium levels leads to activation of the pyruvate dehydrogenase complex (the rate limiting step for carbohydrate oxidation), the increased glucose utilization observed in isolated perfused hearts in the SERCA group may reflect a higher level of mitochondrial calcium.