To investigate the role of CpG sequences in anti-DNA induction, immunization experiments were performed in mice to assess the immunogenicity of native Escherichia coli (EC) and calf thymus (CT) in incomplete Freund's adjuvant. The effects of CpG sequences were further tested by comparing the adjuvant properties of a synthetic phosphorothioate oligonucleotide with a CpG motif to one with a GpC sequence. Both EC and CT DNA alone induced a limited anti-DNA response. For CT DNA, the addition of a CpG ODN significantly enhanced responses whereas for EC DNA, the presence of a CpG oligonucleotide (ODN) or control GpC ODN did not increase responses compared to EC DNA alone. Specificity analysis by ELISA indicated that these immunizations led to the generation of cross-reactive anti-DNA autoantibodies. These results thus extend the adjuvant effects of CpG sequences to self antigens and suggest mechanisms by which self and foreign antigens can interact in the generation of autoimmunity.