Blood-brain barrier (BBB) compromise is a significant pathologic event that manifests early following traumatic brain injury (TBI). Because many signaling cascades are initiated immediately after the traumatic event, we were interested in examining acute differential protein expression that may be involved in BBB function. At acute time points postinjury, altered protein expression may result from altered translation efficiency or turnover rate rather than from a genomic response. The application of tandem 2-D gel electrophoresis and mass spectrometry analysis is a powerful approach for directly screening differential protein expression following TBI. Using comparative 2-D gel analysis, we selected candidate protein spots with apparent altered expression and identified them by mass spectrometry. Cyclophilin A was selected for further analysis because it has been implicated in endothelial cell activation and inflammation, and studies have suggested cyclosporine A, an inhibitor of all cyclophilin isoforms, might be beneficial after TBI. We examined if altered expression of cyclophilin A in the brain vasculature might play a role in BBB function. We found significantly increased cyclophilin A levels in isolated brain microvessels 30 min following injury. Postinjury administration of cyclosporine A significantly attenuated BBB permeability measured 24 hr postinjury, suggesting cyclophilin activity after TBI may be detrimental. However, direct injection of purified recombinant cyclophilin A attenuated both BBB permeability and tissue damage in a stab wound model of injury. These findings suggest that increased expression of cyclophilin A may play a protective role after TBI, whereas other cyclophilin isoforms may be detrimental.