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 -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 coefficientsof 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.