This study investigates the expression, activation, and cellular localization of caspase-3 (CPP32) in the mouse brain following temporary middle cerebral artery occlusion (MCAO). Caspase-3p32 was constitutively expressed in neurons throughout the brain, with higher levels in the piriform cortex. Caspase-like enzyme activity increased in brain homogenates 0–3 hours after reperfusion and peaked at 30–60 minutes. Caspase-3p20 immunoreactivity became prominent in neuronal perikarya within the MCA territory at reperfusion and was detected on immunoblots 1–12 hours later. DNA laddering and TUNEL-positive cells were observed 6–24 hours after reperfusion. Caspase-3p20 was visualized in TUNEL-positive cells, consistent with apoptotic mouse cerebellar granule cells on postnatal day 5. These findings suggest a time-dependent evolution of ischemic injury characterized by the activation of caspase-like enzymes and the subsequent appearance of caspase-3p20, followed by morphological and biochemical features of apoptosis. The study also discusses the regional differences in caspase-3 activation and the potential therapeutic implications of caspase inhibition in ischemic cell death.This study investigates the expression, activation, and cellular localization of caspase-3 (CPP32) in the mouse brain following temporary middle cerebral artery occlusion (MCAO). Caspase-3p32 was constitutively expressed in neurons throughout the brain, with higher levels in the piriform cortex. Caspase-like enzyme activity increased in brain homogenates 0–3 hours after reperfusion and peaked at 30–60 minutes. Caspase-3p20 immunoreactivity became prominent in neuronal perikarya within the MCA territory at reperfusion and was detected on immunoblots 1–12 hours later. DNA laddering and TUNEL-positive cells were observed 6–24 hours after reperfusion. Caspase-3p20 was visualized in TUNEL-positive cells, consistent with apoptotic mouse cerebellar granule cells on postnatal day 5. These findings suggest a time-dependent evolution of ischemic injury characterized by the activation of caspase-like enzymes and the subsequent appearance of caspase-3p20, followed by morphological and biochemical features of apoptosis. The study also discusses the regional differences in caspase-3 activation and the potential therapeutic implications of caspase inhibition in ischemic cell death.