Multi-Western blots of more than 400 proteins were performed from brain extracts of mice submitted to transient focal ischemia induced by 1 h middle cerebral artery (MCA) thread occlusion. Measurements were carried out in groups of six animals in sham-operated controls, at the end of 1 h ischemia, and after 3 and 12 h recirculation. After MCA occlusion up to 45% of proteins were up- or downregulated in the ipsilateral hemisphere by a factor of 1.5 or more, as compared to sham-operated controls. The temporal regulation of several proteins in the ischemia-affected hemisphere after 1 h MCA thread occlusion is described. In the non-ischemic hemisphere the number of regulated proteins was close to 50%, indicating a hitherto unrecognized involvement of the opposite side. The proteomic approach of brain injury analysis goes beyond previous screenings of gene expression at the transcriptional level and although our study provides further evidence for the complexity of multiinjury pathways in the evolution of ischemic brain damage it may help to identify key mediators of ischemic injury.