Calpains are intracellular proteinases whose proteolytic activity is directed mainly against the cytoskeleton and regulatory proteins. We studied the presence of calpain by immunohistochemistry in a rat model of reversible focal cerebral ischemia (3 h) at various times of reperfusion. The numbers of calpain-positive cells on the ischemic side were compared with the non-ischemic side. In controls only 2 +/- 1% cells were positive, whereas the cortex of the ischemic vs the non-ischemic side showed 88 +/- 3% vs 13 +/- 4% calpain-positive cells (p < 0.001), and the basal ganglia 47 +/- 3% vs 13 +/- 4% (p < 0.01) after 3 h ischemia and 24 h reperfusion. This is the first demonstration of elevated intracellular levels of calpains in areas of cerebral ischemia. Longer reperfusion resulted in an increase in calpain positivity.