Monoclonal antibodies were raised to two regions of calpain 3 (muscle-specific calcium-activated neutral protease), which is the product of the gene that is defective in limb-girdle muscular dystrophy type 2A. The antibodies produced characteristic patterns of bands on Western blots: normal calpain 3 protein was represented by bands at 94 kd, plus additional fragments at approximately 60 or 30 kd, according to the antibody used. Specificity was confirmed by the loss of all bands in patients with null gene mutations. The "normal" profile of bands was observed in muscle from 33 control subjects and 70 disease-control patients. Calpain 3 protein was found to be extremely stable in fresh human muscle, with full-size protein being detected 8 hours after the muscle had been removed. Blots of muscle from nine limb-girdle muscular dystrophy type 2A patients with defined mutations showed variation in protein expression, with seven showing a clear reduction in the abundance of protein detected. No simple relationship was found between the abundance and clinical severity. Two patients showed normal expression of the full-size 94 kd band accompanied by a clear reduction in the smaller fragments. This pattern was also observed in one patient with an undefined form of limb-girdle dystrophy. These results indicate that immunodiagnosis is feasible, but caution will need to be exercised with the interpretation of near-normal protein profiles.