Assessment of myocardial infarct size in acute experimental models is usually done by triphenyltetrazolium-chloride (TTC) staining. A certain period of reperfusion is mandatory for discrimination of the infarct zone, especially after relatively short ischemic periods. However, it is unclear what the optimal reperfusion time is for full delineation of the infarct following 30 min of myocardial ischemia in the rabbit. This study compares infarct size, assessed by TTC, in anesthetized open-chest rabbits subjected to 30 min of coronary artery occlusion followed by either 2 (n=14)v4 (n=14) (protocol 1), or 3 (n=8)v6 (n=7) h of reperfusion (protocol 2). Area at risk was assessed by blue dye and necrotic zone by TTC staining. Protocol 1: heart rate and mean blood pressure were comparable in both groups throughout the protocol. Regional myocardial blood flows in both the ischemic and non-ischemic zones during ischemia and after 2 h of reperfusion were comparable between the groups. Regional myocardial blood flow in the post-ischemic zone deteriorated between 2 and 4 h (1.11±0.15v0.58±0.09 ml/min/g, respectively;P=0.0004) of reperfusion. The size of the area at risk was comparable (0.31±0.03v0.33±0.03 of the LV weight in the 2 and 4 h reperfusion groups). However, the ratio of the necrotic zone to the ischemic zone at risk was 63% larger in the 4 compared to the 2 h of reperfusion group (0.31±0.04v0.19±0.03, respectively,P=0.02). Analysis of covariance performed on the weight of tissue that developed necrosis and the weight of ischemic zone at risk revealed a significant effect of the reperfusion time (P=0.014). Protocol 2: there was no difference in infarct size between rabbits subjected to three (0.38±0.05 of the area at risk)v6 h (0.41±0.07) of reperfusion (P=0.72). Analysis of covariance performed on the weight of tissue that developed necrosis and the weight of ischemic zone at risk did not reveal a significant effect of the reperfusion time. Infarct size as assessed by TTC following 30 min of myocardial ischemia, is smaller when measured 2 h after reperfusion than after 4 h of reperfusion. At least 3 h of reperfusion is needed to delineate infarct size by tetrazolium staining following 30 min of ischemia.