For immunotherapy to become more effective, there is a need to maximize the antitumor response at the tumor site as well as to eliminate tumor cell variants that lack a given tumor antigen or the ability to present it. We have previously shown that wild-type (WT) cells from the K1735 melanoma (K1735-WT) are rejected following vaccination with cells (K1735-1D8) transfected to express scFv from the anti-CD137 monoclonal antibody 1D8, and that CD4(+) T cells and natural killer (NK) cells are needed for this rejection. We now show that tumors harvested 4 to 10 days after mice had been transplanted with K1735-1D8 cells or a mixture of K1735-1D8 and K1735-WT cells contained more NK cells and that they had an increased percentage of CD4(+) T lymphocytes producing IFNgamma or tumor necrosis factor-alpha. We further show that the percentage of NK cells was higher in B16-1D8 melanomas expressing anti-CD137 scFv than in the WT tumors and that the percentage of FoxP3(+) cells was lower. Admixture of 10% K1735-1D8 cells prevented the progressive growth of transplanted K1735-WT cells in syngeneic mice and also of cells from the antigenically different sarcoma Ag104. Inhibition of WT tumor cells by tumor cells transfected to express anti-CD137 scFv was shown also with the TC1 carcinoma and B16 melanoma. Furthermore, injection of an adenovirus vector, Ad-1D8, which encodes anti-CD137 scFv into established B16 melanomas, significantly prolonged the survival of tumor-bearing mice and could induce regression. Our data suggest that targeting of anti-CD137 scFv to tumors should be explored for therapy for some human cancers.