We isolated and purified 35 kDa protein from the myocardium of the beagle dog and identified it to be annexin V from partial amino acid sequence determination. It was confirmed that anticanine cardiac annexin V rabbit polyclonal antibody, which was produced using the 35 kDa protein, cross-reacts with annexin V of the myocardium, lung, liver, kidney, and brain of the rat. The localization of cardiac annexin V and the effect of ischemia for 30-180 min in the rat were immunohistochemically studied with the use of the Langendorff perfusion heart. In the normal myocardium, annexin V, accompanied by cross-striation, was observed throughout the cell. In ischemia of 30 min, extracellular leakage of annexin V was observed with uneven staining in the cytoplasm. When the ischemic time exceeded 60 min, annexin V was observed in the cell membrane with a decrease of annexin V in the cytoplasm. Also, extracellular leakage of annexin V was observed prominently. In ischemia for 180 min, almost all the annexin V in the cytoplasm disappeared. These results suggest that the level of ischemia can be estimated from the changes in localization of annexin V.