Objective. To analyze the clinical value of anti-DFS70 antibodies in a cohort of patients undergoing routine antinuclear antibodies (ANAs) testing. Methods. Sera with a dense fine speckled (DFS) indirect immunofluorescence (IIF) pattern from 100 consecutive patients and 100 patients with other IIF patterns were tested for anti-DFS70 antibodies by a novel chemiluminescence immunoassay (CIA) and for ANA by ANA Screen ELISA (both INOVA). Results. Among the 100 patients with a DFS IIF pattern, 91% were anti-DFS70 positive by CIA compared to 3% in the comparator group . The CIA and IIF titers of anti-DFS antibodies were highly correlated (rho = 0.89). ANA by ELISA was positive in 35% of patients with the DFS IIF pattern as compared to 67% of patients with other patterns . Only 12.0% of patients with DFS pattern and 13.4% with DFS pattern and anti-DFS70 antibodies detected by CIA had systemic autoimmune rheumatic disease (SARD). Only 5/91 (5.5%) patients with anti-DFS70 antibodies had SARD and their sera were negative on the ANA Screen ELISA. Conclusion. Although anti-DFS70 antibodies cannot exclude the presence of SARD, the likelihood is significantly lower than in patients with other IIF patterns and should be included in test algorithms for ANA testing.