Pathological activation of the intracellular Ca2+-dependent proteases calpains may be responsible for the neuronal pathology associated with neurodegenerative diseases and acute traumas to the central nervous system. Though calpain activation has been shown definitively in traumatic brain injury (TBI), no studies have investigated calpastatin (CAST), the calpains' endogenous and specific inhibitor, after TBI. The present study examined temporal changes in CAST protein following controlled cortical impact injury in the rat. Western blot analyses of CAST in cortex and hippocampus detected two bands corresponding to molecular weights of 130 kDa [high-molecular-weight (HMW)] and 80 kDa [low-molecular-weight (LMW)]. A modest decrease in the HMW band in conjunction with a significant increase in the LMW band was observed in cortex ipsilateral to the site of impact following TBI. Examination of ipsilateral hippocampus revealed an increasing trend in the LMW band after injury, while no changes were observed in the HMW band. Thus, observable changes in CAST levels appear to occur several hours after reported calpain activation and cleavage of other substrates. In addition, a new analysis was performed on previously published data examining calpain activity in the same tissue samples used in the present study. These data suggest an association between decreases in calpain activity and accumulation of LMW CAST in the ipsilateral cortex following TBI. The present study cannot exclude proteolytic processing of CAST to LMW forms. However, the absence of reciprocity between changes in LMW and HMW bands in consistent with other data suggesting that rat brain could contain different CAST isoforms.