An immunofluorescence microscopy method for following changes in myofibrillar-bound calpain 3 was developed. Afterward, proteolytic changes in calpain 3(p94), calpain 1, titin, and nebulin were examined in myofibrils prepared from ovine longissimusthoracis et lumborum (LTL) stored for 0, 1, 2, and 3 days postmortem. Western blot analysis revealed that the levels of intact calpain 3 (expressed as percentage of the level immediately postmortem) were 80%, 10% and not detectable in myofibrils prepared at 1, 2, and 3 days, respectively. Western blots for calpain 1 also indicated conversion of the intact protein (80 kDa) to a 76 kDa fragment during the same time period. Thus calpains 1 and 3 appear to be activated during postmortem storage. Immunofluorescence microscopy using an IS1 region specific antibody revealed that calpain 3 staining was most intense at the sarcomere Z- and M-lines. The fluorescence intensity declined significantly during storage, paralleling changes in the proteolytic breakdown of titin and nebulin associated with these structures.