Abstract Autoantibodies to calpastatin (endogenous inhibitor of calpain, a calcium-dependent neutral proteinase) have been detected in sera of patients with rheumatoid arthritis (RA) and other diseases. We investigated the epitope reactivity of anticalpastatin autoantibodies in patients with rheumatic diseases. cDNAs encoding each calpastatin domain (L, I, II, III, and IV) were amplified by PCR and ligated into an expression vector. The fusion proteins were expressed in E. coli. The presence of autoantibodies specific for each calpastatin domain was assayed in sera of patients with various rheumatic diseases by immunoblotting the fusion proteins with these sera. Of the RA patient sera, 81% reacted with at least one calpastatin domain. This reaction was significantly greater than with sera from patients with systemic lupus erythematosus (46%), scleroderma (32%), polymyositis/dermatomyositis (43%), and normal controls (13%). Domains I and II were recognized by RA patient sera significantly more than by other patient sera, whereas domains III and IV reacted almost equally among all patient sera. Although, collectively, sera from RA and lupus patients reacted equally with all domains, scleroderma sera tended to react with only domains I and IV and myositis sera tended to recognize only domains III and IV. Patients with RA positive for anticalpastatin antibodies exhibited more active disease (i.e., a higher erythrocyte sedimentation rate and C-reative protein level) than antibody-negative patients. Our results suggest that anticalpastatin antibodies were detected in RA with the highest frequency and that different domain reactivity was shown among different diseases. The presence of these antibodies in sera may be related to the type of disease and, in RA, with disease activity, suggesting their importance in rheumatic disorders.