OBJECTIVE: To compare the autoantigenicity of the recently described N-terminally elongated PM–Scl-75 protein with that of PM–Scl-100 and the originally defined PM–Scl-75 polypeptide, and to determine its value for analyzing sera from patients with the polymyositis (PM)/scleroderma overlap syndrome. METHODS: Serum samples obtained from patients with the PM/scleroderma overlap syndrome and from patients with several other diseases were analyzed for the presence of autoantibodies reactive with recombinant PM–Scl-100 and PM–Scl-75 (both the original and the longer form) proteins, in an enzyme-linked immunosorbent assay (ELISA). RESULTS: Autoantibodies recognizing the longer PM–Scl-75 protein isoform were detected in 28% of the patients with PM/scleroderma. This percentage is slightly higher than that for PM–Scl-100 (25%) and is significantly higher than that for the previously defined PM–Scl-75 protein (11%). In addition, we identified a significant number of patients who had anti–PM–Scl-75 but not anti–PM–Scl-100 antibodies. This finding contrasts with what has been previously reported for the shorter version of the PM–Scl-75 protein. CONCLUSION: Our data indicate that use of the long PM–Scl-75 isoform in addition to PM–Scl-100 in ELISAs significantly increases the number of patients in whom anti–PM-Scl autoantibodies can be detected.