We investigated the role of 4-1BB, a T cell co-stimulatory molecule, in alloimmune responses. In vivo mixed lymphocyte reactions showed that 4-1BB was preferentially expressed on actively dividing CD4(+) and CD8(+) T cells. Furthermore, following alloantigen challenge, the draining lymph nodes contained subpopulations of 4-1BB-expressing CD4(+) and CD8(+) T cells. 4-1BB-deficient C57BL/6 mice showed a delayed rejection of cardiac transplants mismatched for the major histocompatibility complex. Longer transplant survival was induced by blockade of 4-1BB/4-1BB ligand (4-1BBL) interactions using an anti-4-1BBL monoclonal antibody. Histological analysis showed that prolonged transplant survival in the 4-1BB-deficient and anti-4-1BBL-treated mice correlated with reduced lymphocytic infiltration and vasculitis in the donor heart tissue. Taken together, our data suggest that blockade of 4-1BB/4-1BBL interactions inhibited the expansion of alloreactive T cells and reduced CTL activity against host alloantigen, which in turn resulted in the prolongation of allograft survival. Blockade of the 4-1BB co-stimulatory pathway may be useful for preventing allograft rejection.