Transgenic mice are increasingly used to probe genetic aspects of cardiovascular pathophysiology. However, the small size and rapid rates of murine hearts make noninvasive, physiological in vivo studies of cardiac bioenergetics and contractility difficult. The aim of this report was to develop an integrated, noninvasive means of studying in vivo murine cardiac metabolism, morphology, and function under physiological conditions by adapting and modifying noninvasive cardiac magnetic resonance imaging (MRI) with image-guided ³¹P magnetic resonance spectroscopy techniques used in humans to mice. Using spatially localized, noninvasive ³¹P nuclear magnetic resonance spectroscopy and MRI at 4.7 T, we observe mean murine in vivo myocardial phosphocreatine-to-ATP ratios of 2.0 ± 0.2 and left ventricular ejection fractions of 65 ± 7% at physiological heart rates (∼600 beats/min). These values in the smallest species studied to date are similar to those reported in normal humans. Although these observations do not confirm a degree of metabolic scaling with body size proposed by prior predictions, they do suggest that mice can serve, at least at this level, as a model for human cardiovascular physiology. Thus it is now possible to noninvasively study in vivo myocardial bioenergetics, morphology, and contractile function in mice under physiological conditions.