Coronary stenosis results in transmurally non-uniform blood flow with the inner layers of the left ventricular wall typically suffering a more severe hypoperfusion relative to the outer layers. Coupled with numerous other transmural non-uniformities such as systolic tension development and oxygen needs, the heterogeneous blood flow distribution in the presence of coronary stenosis is expected to result in transmurally heterogeneous ischemia. All previous NMR spectroscopy studies of myocardial metabolism and bioenergetics under normal and ischemic conditions treated the organ as a homogeneous tissue. We have utilized spatially localized 31P NMR spectroscopy together with non-NMR measurements of regional blood flow to study the myocardium with transmural spatial differentiation under normal and flow-restricted conditions. 31P NMR and blood flow data obtained concurrently on each heart revealed that sustained coronary artery stenosis resulted in transmurally non-uniform ischemia which largely paralleled the hypoperfusion pattern. The reduction in creatine phosphate content (with consequent elevation of Pi) and hypoperfusion was tightly correlated in the subendocardium for flow rates less than approximately 0.7 mL/min per g wet myocardium. The high energy phosphate and Pi content of the epicardium, however, was responsive not only to the flow to this region but also to the extent of ischemia in the subendocardial layers. These results document the utility of localized NMR spectroscopy in physiologic research and suggest potential biomedical applications due to the tight correlation noted between alterations in blood flow and changes in the phosphorylated metabolite levels detected by 31P NMR.