B1 cells usually show preferential responses to T cell‐independent antigens. To ask whether B1 cells could respond to CD40‐mediated stimulation for proliferation and differentiation, and whether CD40‐mediated signals are involved in the production of autoantibodies by B1 cells, we compared responses to our newly established agonistic anti‐mouse CD40 monoclonal antibody (mAb) between B1 and B2 cells from autoimmune‐prone (NZB X NZW) F1 mice. Stimulation with this mAb induced a similar level of proliferative responses of both B1 and B2 cells, as well as an increase in expression of cell surface molecules I‐A, CD54, CD23, CD80, and CD86. While co‐stimulation with interleukin (IL)‐4 markedly augmented proliferative as well as IgG1 and IgE antibody responses of both B1 and B2 cells, co‐stimulation with IL‐5 augmented proliferative and IgM antibody responses of only B1 cells. Splenic B1, but not B2 cells from young (NZB X NZW) F1 mice spontaneously produced substantial amounts of IgM including IgM anti‐DNA antibodies, and the levels increased in case of stimulation with anti‐CD40 mAb alone, or to a greater extent with the mAb plus IL‐4 and IL‐5. Collectively, these results indicate that splenic B1 cells from autoimmune (NZB X NZW) F1 mice have a comparable responsiveness to the CD40‐mediated stimulation to that of B2 cells, which would be a potent regulatory mechanism involved in the spontaneous production of autoantibodies by B1 cells.