To investigate mechanisms for the induction of anti-DNA antibodies in systemic lupus erythematosus (SLE), the specificity of anti-DNA antibodies was determined in sera from SLE patients and normal control subjects. As a marker of these responses, the reactivity to single-stranded DNA of various mammalian and bacterial species origin was tested by enzyme-linked immunosorbent assay. Patients with SLE demonstrated serum antibodies to all six types of DNA tested, whereas normal control subjects showed appreciable antibody responses only to DNA obtained from Micrococcus lysodeikticus (MC) and Staphylococcus epidermidus (SE). Anti-DNA antibodies in normal sera appeared to recognize unique sites on the DNA because MC DNA failed to inhibit antibody binding to SE DNA, and vice versa; in contrast, SLE antibody binding to MC DNA could be inhibited by SE as well as other DNA, suggesting recognition of a more widely shared epitope. The expression in normal sera of antibodies specific for certain bacterial DNA is consistent with their induction by structural determinants on these DNA molecules that are immunogenic. DNA may therefore represent another bacterial macromolecule capable of inducing cross-reactive antibodies in human autoimmune disease.