Autoantibodies have been used extensively as a useful biomarker in systemic lupus erythematosus and other autoimmune rheumatic diseases. Antinuclear antibodies by immunofluorescence are a standard clinical test to screen for evidence of systemic autoimmunity. Different specific autoantibodies are associated with particular diagnoses, symptoms, unique syndromes, subsets of the disease and clinical activity. They are produced prior to the onset of clinical manifestations and have predictive value. This review focuses on a critical re-evaluation of the clinical significance of autoantibodies. Disease subsets defined by autoantibodies, coexistence of disease marker antibodies, and problems in testing and interpreting results are examined. Clinical approaches in differential diagnosis of antinuclear antibodies and the significance of antinuclear antibodies in healthy individuals are also discussed.