Interleukin (IL)-10 enhances humoral immunity by inhibiting macrophage activation and promoting the development of Th2 cytokine synthesis. In this study we investigated the ability of conventional Ly-1- (CD5-) B cells to produce IL-10 protein. Highly purified normal, naive splenic B cells from both BALB/c and C57BL/6 mice produced IL-10 in response to stimulation with the mitogen LPS. In addition, B cells from antigen primed mice also produced IL-10 upon antigen restimulation in vitro, as analyzed by ELISA and by bioassay. Removal of Ly-1+ B cells did not appreciably reduce IL-10 production, indicating that conventional Ly-1- B cells produced IL-10. These results indicate that normal Ly-1- B cells produce significant quantities of IL-10 during mitogen- or antigen-driven immune responses. The production of IL-10 by conventional B cells may enhance their capacity to promote humoral immune responses.