The purpose of this study was to evaluate flow heterogeneity and impaired reflow during reperfusion after 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 nonischemic controls. Relative flow heterogeneity was expressed as relative dispersion (RD) and computed as standard deviation/mean. In postischemic vs. preischemic 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 considered separately (1.28 ± 0.74 vs. 0.30 ± 0.09 and 0.69 ± 0.22 vs. 0.38 ± 0.08; P < 0.05 for both comparisons, respectively) during early reperfusion. During late reperfusion, the increased RD for the whole LV and Endo remained significant (0.70 ± 0.22 vs. 0.37 ± 0.07 and 1.06 ± 0.55 vs. 0.30 ± 0.09; P < 0.05 for both comparisons, respectively). In addition to the increase in postischemic 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 postischemic flow heterogeneity, substantially reducing the significance of measured global LV reflow. These observations emphasize the need to quantify regional flow during reperfusion after sustained no-flow ischemia in the isolated rabbit heart.