Phosphorus-31 nuclear magnetic resonance spectroscopy can determine the status of high energy phosphates in vivo. However, its application to human cardiac studies requires precise spatial localization without significant contamination from other tissues. Using image-selected in-vivo spectroscopy (ISIS), a technique that allows three-dimensional localization of the volume of interest, 12 subjects were studied to determine the feasibility and reproducibility of phosphorus-31 spectroscopy of the human heart. Nuclear magnetic resonance imaging was performed using a commercial 1.5 tesla system to define the volume of interest. Phosphorus-31 spectra were obtained from the septum and anteroapical region of the left ventricle in 10 studies. Relative peak heights and areas were determined for high energy phosphates. The mean phosphocreatine to adenosine triphosphate ratio was 1.33 +/- 0.19 by height analysis and 1.23 +/- 0.27 by area analysis. Duplicate measurements in four subjects showed a reproducibility of less than or equal to 10% in three of the subjects. All spectra showed significant signal contribution from the 2,3 diphosphoglycerate in chamber red cells without evidence of skeletal muscle contamination. These results demonstrate the feasibility of image-guided phosphorus-31 spectroscopy for human cardiac studies and indicate the potential of this technique to study metabolic disturbances in human myocardial disease.