CpG oligodeoxynucleotides (ODN) are synthetic DNA sequences that mimic bacterial DNA, and bind to the TLR9 receptor. The cells that express TLR9, B cells and dendritic cells, are stimulated by CpG ODN and induce innate and acquired immune responses. Because CpG ODN induce antigen-independent immune activation there has been much interest in the possibility that they may break self tolerance. To test this hypothesis we used a tolerance model with hen egg lysozyme (HEL)-transgenic (Tg) mice, anti-HEL Ig-Tg mice and double (Dbl)-Tg mice injected with CpG ODN alone or together with HEL self antigen. When cultured in vitro, tolerant B cells responded to CpG ODN in a similar way as the non-tolerant Ig-Tg B cells in terms of cell proliferation, NFkappaB activation and CD69 expression. Despite these potent in vitro stimulatory effects of CpG ODN alone, HEL-Tg mice injected with CpG ODN alone, or in combination with low dose antigen (4 microg HEL), surprisingly did not produce any detectable anti-HEL Ab. However, HEL-Tg or Dbl-Tg mice immunized with CpG ODN plus higher doses of self antigen showed strong antigen-specific humoral responses. Surprisingly, control non-CpG ODN also had partial activity for breaking tolerance and inducing autoantibody production when administered in combination with self antigen, though not when used alone. Despite the production of high titers of anti-HEL Ab in the immunized HEL-Tg mice, no evidence of autoimmune disease was detected. We conclude that immunization with CpG or control ODN in the presence of a high dose of exogenous self antigen, but not treatment with ODN alone, can break tolerance to self antigen without inducing autoimmune disease in this system.