Measurements of infarct size by use of tripenyltetrazoliumchloride (TTC) is a widely accepted method used to delineate the extent of myocardial necrosis following coronary occlusion and reperfusion in various animal experiments. There is controversy, however, regarding the optimal reperfusion time for estimating the maximally infarcted area by TTC staining in the rat. We tested six different reperfusion times following 90 minutes of regional myocardial ischemia. Group 1 had 5 minutes of reperfusion (n=6), group 2 had 30 minutes of reperfusion (n=6), group 3 had 1 hour of reperfusion (n=6), group 4 had 2 hours of reperfusion (n=6), group 5 had 3 hours of reperfusion (n=6), group 6 had 4.5 hours of reperfusion (n=6). Risk areas, measured by the use of blue dye, were similar among the 6 study groups. Infarct size as a percent of risk area was 57+/-11% in group 1, 74+/-7% in group 2, 61+/-9% in group 3, 71+/-5% in group 4, 70+/-5% in group 5, and 64+/-9% in group 6 (x+/-standard error [SE]). There was no significant difference in infarct size between the groups. However, prior to 60 minutes of reperfusion, patches of pink and white areas were observed within the risk regions, indicating a more difficult assessment of proper delineation of outer regions of necrotic tissue. For acute assessment of infarct size, reperfusion for 60 minutes or more is optimal because the infarct does appear homogeneous at that time and does not become larger with longer reperfusion periods.