Phosphorothioate oligonucleotides (S-oligos) are nucleic acid derivatives that are commonly used as antisense agents. These compounds, similar to bacterial DNA and CpG oligonucleotides, display a variety of immunological activities in vitro and in vivo. To assess further these activities, the antigenicity of a series of S-oligos was assessed in the solid phase using anti-DNA antibodies from sera of patients with systemic lupus erythematosus. By ELISA, S-oligos bound well to anti-DNA antibodies under the same conditions as calf thymus DNA antigen. The specificity for anti-DNA was established by competition assays showing cross-inhibition of binding by DNA and S-oligos. Reactivity with anti-DNA was observed with S-oligos varying in sequence, suggesting interaction with a conserved determinant not strictly dependent on the bases. Furthermore. in comparison with a phosphodiester oligomer of the same sequence, a phosphorothioate showed dramatically increased activity. These findings indicate that structural features associated with the S-oligo backbone promote specific binding to anti-DNA antibodies and influence the size requirement for antigenicity in the solid phase. These observations thus extend the immunological properties of S-oligos and suggest uses of these compounds in the diagnosis and treatment of autoimmune disease.