Cathepsin B, one of major lysosomal cathepsins, and JNK, a downstream component of Rho kinase (ROCK), are two families of proteases, which play an important role in ischemic cell apoptosis. However, the interrelationship between Cathepsin B and JNK in apotosis has not been examined. In the present study, rats were decapitated at 0, 2, 6, 24, 48 h of reperfusion after 2 h of middle cerebral artery occlusion (MCAO); TUNEL-positive cells appeared in the ipsilateral preoptic region during reperfusion after 2-h MCAO, and gradually increased to a peak of 24 h after reperfusion; Phospho-JNK (p-JNK) immunoreactivity, occurring after Cathepsin B expression, was gradually increased and peaked altogether with Cathepsin B at 6-h reperfusion; Fasudil (5 mg/kg, intraperitoneally), an inhibitor of ROCK, decreased the level of p-JNK and apoptotic neurons, and had no effect on cathepsin B; Immunofluorescent double labeling showed that the colocalization of cathepsin B with p-JNK appeared in the preoptic region at 2, 6, 24, 48 h of reperfusion. These findings indicate that a signal transduction pathway by ischemia-reperfusion is most likely to exist: lysosomal cathepsin B-Rho/Rho kinase pathway-JNK signaling pathway-mitochondrial-dependent intrinsic pathway.