(31)P magnetic resonance spectroscopy (MRS) offers a unique means to noninvasively quantify the major cardiac high-energy phosphates, creatine phosphate (PCr) and adenosine 5'-triphosphate (ATP), that are critical for normal myocardial contractile function and viability. Spatially localized (31)P MRS has been used to quantify the in vivo PCr-to-ATP ratio (PCr/ATP) of murine hearts, including those with pressure-overload hypertrophy induced by thoracic aortic constriction (TAC). To date, there has been no approach for measuring the absolute tissue concentrations of PCr and ATP in the in vivo mouse heart that promise a better understanding of high-energy metabolism. A method to quantify in vivo murine myocardial concentrations of PCr and ATP using an external reference is described, validated, and applied to normal and TAC hearts. This new method does not prolong the scan times in mice beyond those previously required to measure PCr/ATP. The new method renders an [ATP] of 5.0 +/- 0.9 (mean +/- SD) and [PCr] of 10.4 +/- 1.4 micromol/g wet wt in normal mouse hearts (n = 7) and significantly lower values in TAC hearts (n = 10) of 4.0 +/- 0.8 and 6.7 +/- 2.0 micromol/g wet wt for [ATP] (P < 0.04) and [PCr] (P < 0.001), respectively. The in vivo magnetic resonance [ATP] results are in good agreement with those obtained using an in vitro enzyme luminescent assay of perchloric acid extracts of the same hearts. In conclusion, a validated (31)P MRS method for quantifying [ATP] and [PCr] in the in vivo mouse heart using spatial localization and an external reference is described and used to demonstrate significant reductions in not only PCr/ATP but [ATP] in hypertrophied TAC hearts.