CD40 ligand (CD40L) has a great potential as a novel treatment for B-cell lymphoma (BCL). It has previously been demonstrated that a nonvirulent strain of Salmonella typhimurium mutant (ST) can be used not only as a vehicle in oral genetic immunization via the intestinal mucosa, but also as an enhancer of interferon gamma- and tumor necrosis factor alpha-mediated immunity. After confirming that human CD40L can up-regulate expression of Fas, B7-1, and B7-2 molecules on murine BCL cells in vitro, we transfected the human CD40L gene into S typhimurium mutant (ST40L), which was administrated orally to determine whether it was able to prevent the growth of BCL in mice. Expression of human CD40L was confirmed immunohistochemically with protein being detected in the Peyer's patches of mice immunized with ST40L. Moreover, human soluble CD40L had been detectable until 7 to 8 weeks after oral administration of ST40L. Although ST alone exhibited some protective effects, ST40L demonstrated a significantly greater protection against the development of CD40 positive BCL compared with the control. In the surviving mice that had been treated with ST40L, a small and hard nodule was formed at the injection site, which was found to be composed of infiltrating lymphocytes expressing Fas ligand. These results have the potential to be a simple, effective, and above all, safe immune-gene therapy against BCL. (Blood. 2000;95:1258-1263)