Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by antinuclear antibody (ANA) production. ANAs bind to DNA, RNA and complexes of proteins and nucleic acids and are important markers for diagnosis and activity. According to current models, ANAs originate from antigen-driven processes; nevertheless, antibody responses to both DNA and RNA binding proteins display features unexpected in terms of current paradigms for antigenicity. These differences may reflect disturbances in both B and T cells critical for autoreactivity. Clinically, ANA testing has new uses for determining classification as well as assessing eligibility for clinical trials. Studies of patients with established disease show frequent seronegativity. In this setting, seronegativity may indicate a stage of disease called post-autoimmunity in which the natural history of disease or effects of immunosuppressive therapies modifies responses. The new uses of ANA testing highlight the importance of understanding autoantigenicity and developing sensitive and informative assays for clinical assessments.