Targeting CD40, a member of the tumor necrosis factor superfamily, using agonist antibodies (Abs) produces dramatic antitumor effects. Indeed, high-dose intravenous anti-CD40 Ab 'licenses' dendritic cells (DCs) that instruct activated CD8(+) cytotoxic T cells to leave lymph nodes (LNs) and penetrate the mesothelioma tumor microenvironment. However, toxic side effects and the potential of an 'overwhelmed' immune response warrant an alternative approach. In this study, we show that injecting lower doses of anti-CD40 Ab directly into the tumor bed avoided toxic side effects and prolonged survival in 60% of mice, with most cured. Unexpectedly, DCs in tumors and LNs 'disappeared', CD8(+) tumor-specific T-cell numbers and function were not enhanced, and T cells did not infiltrate regressing tumors. CD4(+) or CD8(+) depletion only marginally hindered anti-CD40 Ab efficacy implying another effector mechanism. B-cell numbers significantly increased in tumors, draining LNs and spleens during intratumoral anti-CD40 Ab treatment. CD40 targeting had no effect on splenic B-1 cells, obliterated marginal zone B cells and promoted follicular (FO) B-cell activity. Adoptive transfer of tumor antigen-experienced, CD40-activated B cells, or their immunoglobulin products, which recognized autoantigens on mesothelioma cells, protected against tumor challenge. Finally, studies using B-cell knockout mice showed that successful treatment of established tumors required the presence of B cells. Thus, these data suggest that CD40-activated FO B cells can become an important component of an effective antitumor immune response.