Cardiac-specific transgenesis in the mouse is widely used to study the basic biology and chemistry of the heart and to model human cardiovascular disease. A fundamental difference between mouse and human hearts is the background motor protein: mouse hearts contain predominantly the alphaalpha-myosin heavy chain (MyHC) isozyme while human hearts contain predominantly the betabeta-MyHC isozyme. Although the intrinsic differences in mechanical and enzymatic properties of the alphaalpha- and betabeta-MyHC molecules are well known, the consequences of isozyme shifts on energetics of the intact beating heart remain unknown. Therefore, we compared the free energy of ATP hydrolysis (|DeltaG( approximately ATP)|) determined by (31)P-NMR spectroscopy in isolated perfused littermate mouse hearts containing the same amount of myosin comprised of either >95% alphaalpha-MyHC or approximately 83% betabeta-MyHC. |DeltaG( approximately ATP)| was approximately 2 kJ mol(-1) higher in the betabeta-MyHC hearts at all workloads. Furthermore, upon inotropic challenge, hearts containing predominantly betabeta-MyHC hearts increased developed pressure more than alphaalpha-MyHC hearts whereas heart rate increased more in alphaalpha-MyHC hearts. Thus, hearts containing predominantly the betabeta-MyHC isozyme are more energy efficient than alphaalpha-MyHC hearts. We suggest that these fundamental differences in the motor protein energy efficiency at the whole heart level should be considered when interpreting results using mouse-based cardiovascular modeling of normal and diseased human hearts.