Proton NMR techniques were used to characterize acutely ischemic myocardial tissue from the dog. Ligation of the left anterior descending coronary artery for 30 min resulted in a consistent prolongation of the proton spin-lattice relaxation times (T1) in samples from regionally ischemic heart muscle when compared with T1 values for nonischemic areas from the same hearts. The relative prolongation of relaxation times in ischemic tissue was found to increase as the duration of ischemia was extended to 60 or 120 min. T1 values for ischemic tissue were not directly related to tissue levels of high-energy compounds, lactate, or hydrogen ions but largely reflected the increased water content of the regionally ischemic myocardium. Proton NMR analysis provides a means of identifying acute regional ischemia in heart tissue, and in the future may permit three-dimensional imaging of the heart in vivo.