The role of anions in the initiation of ischemia- and reperfusion-induced arrhythmias is unknown. We examined the antiarrhythmic effects of isotonic substitution of extracellular Cl−with NO3−by using the rat Langendorff preparation (n =12 per group). During 30 minutes of regional ischemia, the incidence of ventricular fibrillation (VF) was reduced from 50% in hearts perfused with control solution (containing a Cl−:NO3−ratio of 100:0) to 25%, 0% (p<0.05), 0% (p<0.05), and 0% (p<0.05) by perfusion with solution containing Cl−:NO3−ratios of 75:25, 50:50, 25:75, and 0:100, respectively. The incidence of reperfusion-induced VF was also reduced from 58% to 25%, 8% (p<0.05), 8% (p<0.05), and 0% o(p<0.05), respectively. Similar effects were produced in hearts reperfused after briefer durations of ischemia (10 or 15 minutes). Substitution of NO3−for Cl−also facilitated spontaneous termination of VF. Heart rate and occluded zone size were not affected by anion manipulation. Coronary flow was affected by NO3−, but changes did not correlate with arrhythmias. During ischemia, electrocardiographic changes indicative of class III activity (widening of the ventricular complex) were produced by anion substitution. These changes occurred selectively in the ischemic tissue with no significant influence before ischemia onset. However, the relation between this effect and arrhythmia reduction was not linear and a cause-effect relation is therefore unlikely. In separate groups of hearts (n= 12 per group), switching from 100:0 to 0:100 Cl−:NO3−solution or vice versa 10 seconds after coronary occlusion or just before reperfusion demonstrated that 1) protection against ischemia-induced VF resulted partly from an action in the ischemic zone and partly from an action in the nonischemic zone, and 2) protection against reperfusion-induced VF resulted principally from an action occurring during reperfusion and within the reperfused tissue. To assess whether benefit was offset by deleterious effects on contractile function in nonischemic tissue, we constructed Starling curves in isolated rat hearts. The 0:100 Cl−: NO3−solution had no effect on compliance or contractility at physiological end-diastolic pressures but reduced the slope of the peak systolic pressure-volume relation by ∼20% as end-diastolic pressure was increased above 10 mm Hg. In conclusion, anions appear to play a hitherto unrecognized role in arrhythmogenesis in ischemia and reperfusion. Manipulation of anion homeostasis may represent a novel target for antiarrhythmic drug development.