The effects of regional myocardial ischemia and reperfusion on magnetization transfer (MT) contrast were investigated in an ex vivo perfused piglet heart model. The extent of the ischemic area was defined with perfusion magnetic resonance (MR) studies performed with use of extracellular contrast agents. Relative MT contrast was calculated for a total of 106 regions of interest in nine hearts. In the areas defined as being severely ischemic in the perfusion studies, a small but significant increase in the MT contrast of 18% ± 9 (standard deviation) (n = 35) was found as early as 10 minutes after the start of ischemia. This contrast difference was reduced to 11% ± 10 after 2 hours of total occlusion. The contrast between normal and ischemic tissue can be explained in part by the effect of inflowing blood, which leads to changes in both equilibrium magnetization and the apparent T1 of the perfused tissue. However, theoretical estimation suggests that these flow-related changes would produce a maximal relative change in MT contrast of approximately 4%. The most likely explanation for the rest of the observed changes is alteration in the distribution of cellular water related to the so-called intracellular edema that is known to be associated with the acute phase of myocardial ischemia.