An antibody specific for the calpain isoform m-calpain was used to resolve conflicting results from several studies on the possible role of m-calpain in the pathogenesis of Alzheimer's disease (AD). Levels of the enzyme in both cytosolic and membranous fractions of brain tissue were determined by Western blot analysis. We also demonstrated changes in m-calpain molecules in AD brains using high-resolution 2D gel electrophoresis (2DE). The levels of the m-calpain isoform detected in the cytosolic fraction were significantly increased in AD brains when compared with the levels in controls. On 2DE, m-calpain molecules resolved into eight main spots. These spots were detected in AD brains as well as in control brains, suggesting that the calpain molecule was not qualitatively changed in AD. Quantitative analysis of the m-calpain spots on 2DE, on the other hand, indicated that the ratio of the intensity of four protein spots in the acidic region to that of the total spots was increased in AD brains.