Using the isolated perfused rat heart with transient (10-minute) regional ischemia induced by coronary artery ligation, we have shown that PBN (N-tert-butyl-α-phenylnitrone), an organic spin trap agent designed specifically to form “stable” adducts with free radicals in electron spin resonance studies, can dramatically reduce the vulnerability of the myocardium to reperfusion-induced ventricular fibrillation. Studied in the concentration range of 5-1,000 μM/L, PBN added to the perfusate 5 minutes prior to ischemia exerted a dose-dependent protective effect. At the optimum concentration of 30 μM/L PBN reduced the incidence of ventricular fibrillation to 50% (6 of 12) from its control value of 100% (12 of 12). The antiarrhythmic effect was achieved without any substantial effect on coronary flow or heart rate. Investigating whether this was a direct antiarrhythmic effect, operating during reperfusion, or an indirect effect arising from the action of PBN on the heart during ischemia, PBN (30 μM/L) was added to the perfusion fluid 2 minutes before reperfusion. In the control group, 100% of the hearts fibrillated whereas only 50% fibrillated in the PBN group. Additional studies were designed to ascertain whether the drug caused an absolute reduction in vulnerability to reperfusion-induced arrhythmias (irrespective of the duration of ischemia) or whether it only shifted the ischemic time-reperfusion vulnerability curve to the right (i.e., delayed the onset of vulnerability). Thus, studies were undertaken to define the relation between the duration of ischemia and the incidence of reperfusion-induced arrhythmias in control hearts and hearts treated with PBN. Hearts (12 for each group) were subjected to 5, 10, 20, 30, or 40 minutes ischemia; PBN (30 μM/L) was added to the perfusate either 5 minutes before ischemia or 2 minutes before reperfusion. The results were compared to PBN-free control hearts. In each instance, a bell-shaped time-response profile was obtained. In the PBN-free controls, this gave a maximal vulnerability to reperfusion-induced arrhythmias after 10 minutes of ischemia; in the PBN-treated hearts, this curve was shifted to the right and slightly downwards. These results indicate that the primary action of PBN is to exert a delaying effect, which essentially extends the duration of ischemia that can be tolerated before the heart becomes highly vulnerable to reperfusion-induced arrhythmias. However, this effect is achieved during the reperfusion period and not during the preceding period of ischemia. The precise mechanism by which this free radical spin trapping agent achieves this unusual protective effect remains to be resolved.