We determined whether spatial distributions of substrate uptake are heterogeneous within the area at risk during reperfusion. Quantitative autoradiography with imaging plates and two long-lived radioisotopes was applied to 15 open-chest, anesthetized rats subjected to 30 min of coronary artery ligation and 30 min of reperfusion. Regions showing increased beta-methyl-[1-14C]heptadecanoic acid ([14C]BMHDA) uptake (166 +/- 17% of that in the nonischemic area) appeared at the lateral borders and subepicardial layer within the area at risk, and 2-deoxy-D-[1-3H]glucose ([3H]DG) uptake was 103 +/- 24% in these regions. Regions with decreased [14C]BMHDA uptake (28 +/- 11%) occupied the midmyocardial layer except at the lateral borders within the area at risk, and [3H]DG uptake was 62 +/- 18% in these regions. The percentage interregional coefficients of variation (index of heterogeneity) in [14C]BMHDA uptake, [3H]DG uptake, and blood flow were higher in the area at risk than in the nonischemic area (76 +/- 23 vs. 21 +/- 7%, 39 +/- 10 vs. 21 +/- 7%, and 49 +/- 19 vs. 14 +/- 4%, respectively). Heterogeneous distributions of substrate uptake may explain the conflicting results concerning substrate metabolism during reperfusion.