We have established a focal preconditioning (PC) paradigm that produces significant and prolonged ischemic tolerance (IT) of the brain to subsequent permanent middle cerebral artery occlusion (MCAO). PC using 10 min of MCAO induces brain tolerance at 1-7 days of reperfusion that requires active protein synthesis. The protective protein(s) involved are unknown. In these studies the increased transcription and translation of the inducible 70-kDa heat shock protein (Hsp70) and the 27-kDa heat shock protein (Hsp27), and astrogliosis/glial fibrillary acidic protein (GFAP) were determined by Northern analysis and immunohistochemistry following PC. Cellular localization of proteins was determined by double labeling. PC produced no brain injury but did increase Hsp70 mRNA transiently at 6 h and increased Hsp27 mRNA later at 24 h for at least 5 days. Protein expression induced by PC exhibited a similar profile. Hsp70 protein was primarily expressed in neurons from 1 to 5 days post-PC throughout the PC cortex. Hsp27 protein expression was initiated later for a much longer period of time. A remarkable astroglyosis was verified with increased astrocytic Hsp27 from 1 to 7 days after PC. Gliosis with increased Hsp27 in the PC cortex was still present but reduced 4 weeks after PC. Therefore, PC that results in brain tolerance/neuroprotection increases neuronal Hsp70 in the PC cortex and activated astrocytic Hsp27 in the PC cortex in a temporal fashion associated with developing IT. The short duration of benign ischemia (PC) that produces IT produces a robust, long-lived cellular and protein synthetic response that extends throughout the entire cortex (i.e. well beyond the MCA perfusion territory). The resulting IT is associated with changes in astrocyte-activation that might provide increased support and protection from injury. Although both Hsp70 and Hsp27 may participate in the neuroprotection/brain tolerance induced by PC, the temporal expression patterns of these proteins indicate that they are not solely responsible for the tolerance to brain injury.