DNA nick end-labeling (TUNEL) and heat shock protein (HSP)70 immunocytochemistry were performed on the same brain sections 1 (n = 6), 3 (n = 12), and 7 (n = 7) days following permanent middle cerebral artery (MCA) occlusions produced in adult rats using the endovascular carotid suture method. In the cortex at 1 and 3 days following MCA occlusions, HSP70 immunoreactive neurons were located outside areas of infarction and showed little evidence of DNA fragmentation. HSP70-stained cortical neurons were intermingled with TUNEL cells near the infarct, but extended for greater distances away from the infarct. DNA fragmentation occurred in CA1 hippocampal neurons in 39% of the animals at 1 and 3 days following ipsilateral MCA occlusion. Bilateral DNA fragmentation occurred in CA1 neurons in one subject. HSP70 protein was expressed in CA1 hippocampal neurons in nine of 18 (50%) animals at 1 and 3 days following MCA occlusions, including all animals that exhibited hippocampal DNA fragmentation. Three animals had bilateral expression of HSP70 in CA1 neurons. Cells that stained for either HSP70 protein or DNA fragmentation existed in close proximity to one another. Approximately 5-7% of HSP70-stained cells were TUNEL stained and 3% of TUNEL-positive cells also stained for HSP70. There was no HSP70 staining or DNA fragmentation in the brains of sham-operated controls (n = 4) or in the brains of animals 7 days following MCA occlusions. These data suggest that ischemic cells capable of translating HSP70 protein generally do not undergo DNA fragmentation. These data support the concept that most HSP70 protein-containing neurons in the cortical "penumbra" and hippocampus survive ischemic injury and are "reversibly injured." It is shown that CA1 hippocampal pyramidal neurons die or are reversibly injured in approximately 50% of animals following permanent MCA occlusions. Although the mechanism of this hippocampal injury is unknown, it could relate to transynaptic activation of N-methyl-D-aspartate (NMDA) receptors that mediate induction of early genes in hippocampus.