A membrane cytoskeletal protein, fodrin, is a substrate for a Ca2+‐dependent protease, calpain. It remains unknown whether μ‐calpain or m‐calpain is involved in the proteolysis of either α‐ or β‐fodrin and in what subcellular localization during ischemia and reperfusion of the brain. To address these issues, we examined the distribution of fodrin and calpain and the activities of calpain and calpastatin (endogenous calpain inhibitor) in the same subcellular fractions. Rat forebrain was subjected to ischemia by a combination of occlusion of both carotid arteries and systemic hypotension, whereas reperfusion was induced by releasing the occlusion. Immunoblotting, activity measurement, and casein zymography did not detect the presence of μ‐calpain or a significant change of m‐calpain level after ischemia or reperfusion. However, casein zymography revealed a unique Ca2+‐dependent protease that was eluted with both 0.18 and 0.40 M NaCl from a DEAE‐cellulose column. α‐ and β‐fodrins and m‐calpain were found to be rich in the synaptosomal, nuclear, and cytosolic subfractions by immunoblotting analysis. Reperfusion (60 min) following ischemia (30 min) induced selective proteolysis of α‐fodrin, which was inhibited by a calpain inhibitor, acetylleucylleucylnorleucinal (400 µM, 1 ml, i.v.). The μ‐calpain‐specific fragment of β‐fodrin was not generated during ischemia‐reperfusion, supporting the possibility of the involvement of m‐calpain rather than μ‐calpain in the α‐fodrin proteolysis.