The lupus susceptibility interval Sle3/5 confers responsiveness to prolactin in C57BL/6 (B6) mice and hyperprolactinaemia induces a lupus-like phenotype in B6.Sel3/5 mice. In this study, the immunostimulatory effects of prolactin in B6 mice containing the Sle3 portion of the Sel3/5 interval (B6.Sle3 mice) were dissected. Because of the Sle3 interval's involvement in activation of myeloid cells, the effect of dendritic cells (DCs) from prolactin-treated B6.Sle3 mice on the phenotype of B6 mice was also evaluated. B cells from prolactin-treated B6 and B6.Sle3 mice and from B6 recipients of prolactin-modulated DCs from B6.Sle3 mice were tested for DNA-reactivity and resistance to B cell receptor (BCR)-mediated apoptosis. The expression of co-stimulatory molecules on lymphocytes and myeloid cells was also evaluated. In prolactin-treated B6.Sle3 mice, transitional type 2 B cells increased while type 1 B cells decreased as a consequence of prolactin-induced resistance to BCR-mediated apoptosis leading to the survival of DNA-reactive B cells. Follicular B cells from prolactin-treated mice expressed increased levels of CD40, B7·2 and IAb, and DCs and monocytes had higher levels of CD44 and B7·2 than placebo-treated mice. Adoptive transfer of DCs from prolactin-treated B6.Sle3 mice to B6 recipients demonstrated the intrinsic ability of prolactin-modulated DCs to induce a development of lupus-like characteristics in B6 mice. Based on these results, prolactin accelerates the breakdown of immune tolerance in B6.Sle3 mice by promoting the survival, maturation and activation of autoreactive B cells, DCs and macrophages.