Copper labeled diacetyl-bis(N4-methylthiosemicarbazone) (Cu-ATSM) is a promising agent for the imaging of hypoxic tissues. In the present study 64Cu(t1/2 = 12.8 h) labeled Cu-ATSM was used in combination with 11C (t1/2 = 20.3 min) labeled acetate as a regional perfusion marker to visualize hypoxic rat heart tissue in an acute left anterior descending (LAD) coronary artery occluded rat model using an ex vivo tissue slice imaging technique. 64Cu-ATSM was injected intravenously c.a. 10 min after occlusion and rats were sacrificed by cervical dislocation 10 min after injection. Carbon-11-acetate was injected 1 min before sacrifice to obtain a measure of blood flow. The heart was dissected, frozen, and cut into 1-mm thick slices with a gauged slicer, and 11C images were obtained with an electronic autoradiography instrument. After decay of 11C, 64Cu images were obtained in the same manner. In ischemic regions, where there was low 11C accumulation, 64Cu showed high accumulation when compared with normal regions. In rats with a large occlusion, the center of the ischemia did not show any accumulation of either 11C or 64Cu, indicating no blood supply. Cu-ATSM appears to be useful for the detection of hypoxia with contrast being observed at short times (10 min) postinjection.