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Objective: Anti-RNA polymerase I/III (anti-RNAP I/III) antibodies are clinically useful markers of scleroderma, and their presence is associated with diffuse skin disease and an increased risk of cardiac and kidney involvement. Although RNAP I antibodies localize to the nucleolus, nucleolar staining by many anti-RNAP antibody-positive sera is not always observed. Nucleolar staining by anti-RNAP antibody-positive sera was examined by double staining with antifibrillarin antibodies to evaluate whether nucleolar staining can be used as a screening test for anti-RNAP I/III antibodies. In addition, the relationships between nucleolar staining and levels of anti-RNAP III antibodies were examined by enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation (IP) assay., Methods: Sera were tested using immunofluorescent antinuclear antibodies on HEp-2 cell slides, by anti-RNAP III ELISA, and by IP assay using (35)S-labeled K562 cell extract. Nucleolar staining by anti-RNAP antibody IP-positive sera was confirmed by double staining using antifibrillarin monoclonal antibodies. The levels of anti-RNAP III antibodies were quantitated by ELISA and by IP assay using a serially diluted reference serum as a standard, and their relationship was analyzed., Results: All 18 anti-RNAP I/III antibody-positive sera showed nuclear speckled patterns, but nucleolar staining was readily noticeable in only 44% of the sera. A positive correlation was found between ELISA and IP units for anti-RNAP III antibodies. The levels of anti-RNAP III antibodies and anti-RNAP I antibodies correlated well, with the exception of a few sera. Levels of anti-RNAP III antibodies were low in sera with nucleolar staining, whereas several sera with high levels of anti-RNAP I antibodies clearly showed nucleolar staining., Conclusion: Although some sera positive for anti-RNAP I/III antibodies clearly stain nucleoli, nucleolar staining is inconsistent and cannot be used to screen for anti-RNAP I/III antibodies.