CD40-ligand (CD40L), a member of the TNF family, is expressed transiently on activated CD4-positive T cells and mediates cognate interaction between T cell and antigen-presenting cell (APC) such as dendritic cells. We and other investigators have reported previously that transduction of human leukemia cells with adenovirus encoding full-length CD40-ligand resulted in upregulation of immune costimulatory molecules, enhance APC activity and generation of CTL to leukemia B cells. However, CD40L is cleaved to a soluble form (sCD40L) by metalloproteases and high levels of sCD40L may contribute to the systemic inflammatory diseases including systemic lupus erythematosus and rheumatoid arthritis, suggesting a potentially deleterious side effect of CD40L gene therapy. In this study we generated a non-cleavable mutant of CD40L to develop a potentially less toxic molecule for CD40L gene therapy. Four mutants of human CD40L (termed CD40Lm1, m2, m3 and m4) with point mutation of amino acids from E112 to P120 (suggested cleavage site) were created by RT-PCR and cloned into retrovirus and adenovirus vectors. These four mutants of CD40L were transduced into tumor cells and assessed sCD40L production by ELISA, demonstrating that all four mutants resulted in a fully non-cleavable mutant of CD40L. We also confirmed that CD40L mutants could stimulate CD40-positive B and dendritic cells and induce phenotypic alterations and IL-12 production. In order to examine systemic side effect of CD40L, we transplanted tumor cells expressing wild-type (CD40Lwt) or non-cleavable mutant of CD40L (CD40Lm3) in nude mice and have observed for one month period. Two weeks after transplantation, mice with tumors expressing CD40Lwt exhibited arthritis, systemic edema and slight diarrhea, but CD40Lm3 did not induce any systemic inflammatory effect. We also found increased plasma levels of sCD40L (>800 pg/ml) in mice transplanted with CD40Lwt transfectant but not in CD40Lm3 transplanted mice. Additionally, mice with CD40Lwt resulted in increased number of infiltrating mononuclear cells in the liver and kidney, whereas no inflammatory cells were observed in the liver of mice with CD40Lm3. Overall, non-cleavable mutant of CD40L is fully capable of inducing immune response with less toxic molecule and useful tool for CD40L gene therapy of leukemia and lymphoma.