A novel [99m]technetium-labeled nitroimidazole was preferentially taken up and retained by hypoxic cardiac muscle. In rat hearts perfused with O2 or N2 equilibrated cell-free medium, uptake of the infused nitroheterocycle and its subsequent washout displayed biphasic kinetics. For both uptake and washout, the early phase was very rapid whereas the late phase was much slower. The amount of radioactivity retained after 40 min of clearance was about two-fold greater in hypoxic hearts than in normoxic hearts. Cardiac myocytes and mitochondria isolated from rat heart also accumulated the nitroheterocycle. Association of the compound with heart cells was inversely related to the level of available oxygen and was independent of intracellular energy level or mitochondrial redox state in the presence of oxygen. The results indicate that this [99mTc]labeled nitroimidazole may serve as a sensitive marker of hypoxic myocardium.