The reduced thiol pool of myocardial tissue represents an important defense mechanism against oxygen toxicity. Since the ischemia-induced depletion of this pool might favor the cytotoxicity of oxygen-derived free radicals produced during reperfusion, we assessed the effects of the thiol group donor N-acetylcysteine in an isolated buffer-perfused rat heart model of ischemia/reperfusion. Fifty hearts were studied. A first series of experiments that consisted of two groups (n = 10) was designed to simulate the conditions of standard cardioplegic arrest. Hearts were subjected to 180 minutes of cold (15° to 18° C) global ischemia and 1 hour of reperfusion. The control group received crystalloid hyperkalemic cardioplegic solution given every 30 minutes during arrest, and the treated group received the same solution supplemented with N-acetylcysteine (0.04 mol/L). On the basis of comparisons of postreperfusion left ventricular developed pressure, maximal dP/dt, and diastolic pressure, N-acetylcysteine-containing cardioplegic solution afforded significantly better protection. A second series of experiments was then undertaken to assess the effects of N-acetylcysteine in hearts subjected to the sequence of ischemic events that is inherent in transplantation procedures. Hearts were cardioplegically arrested, stored for 5 hours at 2° C, subjected to 1 additional hour of ischemic arrest at 15° to 18° C, and reperfused for 60 minutes. Three groups (n = 10) were studied that differed by the modalities of cardioplegic preservation used during the poststorage ischemic interval. One group received multidose unmodified cardioplegic solution. A second group received multidose cardioplegic solution supplemented with N-acetylcysteine (0.04 mol/L), and the third group was given only a single dose of N-acetylcysteine-enriched (0.07 mol/L) cardioplegic reperfusate at the end of arrest. Multidose N-acetylcysteine- containing cardioplegic solution resulted in a significantly better hemodynamic recovery than unmodified cardioplegic solution. The protection afforded by N-acetylcysteine was lost when the drug was given only at the time of reperfusion. We conclude that supplementation of cardioplegic solution with N-acetylcysteine markedly improves postarrest recovery of function, presumably through an enhancement of the reduced thiol pool, which increases the capacity of reperfused myocardium to handle the postischemic burst of free radical production. The clinical relevance of these findings stems from the fact that thiol-containing drugs are available for human use.