Studies employing casein zymographic assays analyzed the effects of varying pH (from pH 6.8 to pH 8.0) on changes in mu-calpain and m-calpain activity in naive, sham-injured and injured rat cortex 3 h following unilateral cortical impact injury. Mu-calpain activity following cortical impact injury was enhanced between pH values of 7.2 and 7.8, with pH 7.5 being optimal. m-Calpain activity was readily detected only between pH values of 7.2 and 7.4, with pH 7.3 producing the most prominent proteolytic activity. These observations suggest that strict control of pH is an important consideration in assessments of brain pH activation by casein zymography. Moreover, activation of different calpain isoforms, especially after traumatic brain injury, may be differentially influenced by smaller changes in physiological pH than previously recognized.