The phenotype of mouse thymic B cells and their capacity to induce T cell negative selection in vitro were analyzed. Thymic B cells expressed B cell markers such as IgM, Fc gamma receptor, CD44, heat-stable antigen, LFA-1 and CD40. In addition, they were positive for the activation molecule CD69 and displayed high levels of B7-2. Although thymic B cells expressed CD5 on their surface, no CD5-specific mRNA was detected. Moreover, thymic B cells induced a stronger deletion of TCR-transgenic (TG) thymocytes than splenic B cells, which had low CD69 and B7-2 levels. Interestingly, CD40-activated splenic B cells up-regulated CD69 and B7-2 and acquired a capacity to induce T cell deletion comparable to that of thymic B cells. Moreover, thymic B cells from CD40-deficient mice displayed lower CD69 and B7-2 levels than control thymic B cells, and lower capacity to induce the deletion of TCR TG thymocytes. These results support the hypothesis that CD40-mediated activation of thymic B cells determines a high efficiency of antigen presentation, suggesting that within the thymus B cells may play an important role in the elimination of autoreactive thymocytes.