The purpose of this study was to evaluate flow heterogeneity and impaired reflow during reperfusion following 60 min global no-flow ischemia in the isolated rabbit heart. Radiolabeled microspheres were used to measure relative flow in small left ventricular (LV) segments in five ischemia + reperfused hearts and in five non-ischemic controls. Relative flow heterogeneity was expressed as relative dispersion (RD) and computed as standard deviation/mean. In post-ischemic vs. pre-ischemic hearts, RD was increased for the whole LV (0.92± 0.41 vs. 0.37 ± 0.07,P < 0.05)) as well as the subendocardium (Endo) and subepicardium (Epi) considered separately (endo: 1.28 ± 0.74 vs. 0.30 ± 0.09; epi: 0.69 ± 0.22 vs. 0.38 ± 0.08; P < 0.05 for both comparisons) during early reperfusion. During late reperfusion, the increased RD for the whole LV and subendocardium remained significant (LV: 0.70 ± 0.22 vs. 0.37 ±0.07; endo: 1.06 ± 0.55 vs. 0.30 ± 0.09;P < 0.05 for both comparisons). In addition to the increase in post-ischemic flow heterogeneity, there were some regions demonstrating severely impaired reflow, indicating that regional ischemia can persist despite restoration of normal global flow. Also, the relationship between regional and global flow was altered by the increased post-ischemic flow heterogeneity, substantially reducing the significance of measured global LV reflow. These observations emphasize the need to quantify regional flow during reperfusion following sustained no-flow ischemia in the isolated rabbit heart.