Calpain, a calcium-activated neutral protease family, has been implicated in the neuropathologic sequelae accompanying various neurological disorders. We have characterized the distribution and time course of calpain activation following brain injury in the rat, using a monoclonal antibody that recognizes calpain-generated breakdown products (BDPs) of spectrin. Adult male Sprague-Dawley rats received lateral fluid percussion brain injury of moderate severity (2.2–2.4 atm, n = 35) or served as controls (uninjured, n = 12). One group of animals (n = 21) were sacrificed at either 30 minutes (min), 1 day, or 3 days post-injury, and selected brain regions were prepared for Western blot analysis. The remaining animals (n = 26) were sacrificed at 90 min, 4 hours (h), 1 day, or 7 days post-injury, and immunohistochemistry was performed. Spectrin BDPs were found predominantly in the hemisphere ipsilateral to the injury site, located primarily in cortical and hippocampal regions which exhibit neuronal death. Calpain-mediated spectrin breakdown was detected at 90 min in dendrites and axons, and by 4 h in neuronal perikarya. By 1 day post-injury, cortical and hippocampal regions of calpain activation had increased in size. Delayed spectrin breakdown was observed in the thalamus, both at 3 days and 7 days after injury. These results suggest that calpain may play an important role in the neurodegenerative process following brain injury.