Objective: The adenine nucleotide translocase (ANT) facilitates the exchange of mitochondrial ATP and cytosolic ADP over the inner mitochondrial membrane, supplying the cell with energy. Myocardial overexpression of ANT1 leads to an accelerated mitochondrial ATP/ADP transport in transgenic rat hearts. Myocardial Ca²⁺ homeostasis and contraction are the largest ATP consumers in myocytes and may significantly benefit from an accelerated mitochondrial ATP supply. Methods and Results: To explore the effect of an advanced mitochondrial ATP/ADP transportation on Ca²⁺ homeostasis, oxalate-supported ⁴⁵Ca²⁺ uptake by the cardiac sarco(endo)plasmic reticulum (SR) Ca²⁺ ATPase (SERCA2a) was measured in left ventricular homogenates. Compared to wild type (WT), the ANT1 transgenic rats revealed an elevation in Ca²⁺ transport (33.0±11.5%; p<0.01) into the SR and as quantified by western blot analysis, a significantly higher SERCA2a protein level (22.2±4.7%; p<0.01). The plasmalemmal Ca²⁺ ATPase (PMCA) indicated an advanced protein expression (23.8±4.8%; p<0.01), whereas protein amount of Na⁺/Ca²⁺ exchanger was not altered in ANT1 overexpressing rats. ANT1 transgenic rats showed morphologic, but no pathophysiologic changes in left ventricular chamber size and a distinct increase in sarcomere length. Quantification of sarcomere proteins exhibited a significant elevation of actin (33.2±9.3%; p<0.03), heavy chain cardiac myosin (42.5±14.8%; p<0.05) and troponin I protein (51.5±13.6%; p<0.01). Conclusions: Overall, these data reveal a close association of accelerated mitochondrial ATP transport with increased expression of contractile components and elevated Ca²⁺ transport into the SR and over the sarcolemma. Thus, advanced ATP supply seems to be converted into elevated Ca²⁺ transport- and cellular work-reserve.