Estrogen can modulate autoimmunity in certain models of systemic lupus erythematosus. Recently, we have shown that it can mediate survival and activation of anti-DNA B cells in a mouse transgenic for the heavy chain of a pathogenic anti-DNA antibody. To identify whether estrogen effects reflect increased prolactin secretion, we characterized B-cell autoreactivity in transgenic mice given both bromocriptine (an inhibitor of prolactin secretion) and estradiol. Treatment of mice with estradiol plus bromocriptine led to reduced titers of anti-DNA antibodies and diminished IgG deposition in kidneys compared with treatment with estradiol alone. However, mice treated with estradiol plus bromocriptine showed an expansion of transgene-expressing B cells and enhanced Bcl-2 expression, similar to those of estradiol-treated mice. We identified anergic high-affinity anti-DNA B cells in mice treated with estradiol plus bromocriptine, and we showed by molecular analysis of anti-DNA hybridomas that their B cells derive from a naive repertoire. Thus, the estradiol-induced breakdown in B-cell tolerance can be abrogated by bromocriptine, which induces anergy in the high-affinity DNA-reactive B cells. These studies demonstrate that some of the effects of estrogen on naive autoreactive B cells require the presence of prolactin and, thus, suggest potential therapeutic interventions in lupus.