We have employed the DNA-mediated gene transfer method to introduce the allogenic major-histocompatibility-complex(MHC)-class-I gene H-2Kb into the K36.16 tumor cells, H-2k, in order to generate tumor-specific immunity. The acquisition of immunogenicity by the H-2Kb-transformed clones following gene transfer is associated with the reduction of constitutive reactive superoxide radicals. When the levels of cellular superoxide for the H-2Kb-positive immunogenic clones were determined, they were significantly lower (30 to 60%) than that of the parental K36.16 tumor cells. This reduction of superoxide in the H-2Kb-transformed cells was associated with a significant increase in the level of Cu-Zn superoxide dismutase (SOD) and GPX I, together with a reduction in the DNA-binding form of the NF-kappa B transcription factor. The K36.16 parental tumor cells were also found to be relatively more resistant to the cytotoxic effects of hydrogen peroxide in vitro. To further support the role of superoxide anion radicals in tumorigenesis, in vivo depletion of glutathione promoted the tumorigenicity of the H-2Kb-transformed clones in (AKR/J x C57BL/6/J) F1 mice, whereas SOD was able to reduce their tumorigenicity. In addition, the presence of R-sulfoxine (BSO) in spleen-cell cultures in vitro abolished the ability of the immune lymphocytes to develop into tumor-specific cytotoxic T lymphocytes (CTL). These observations support the concept that oxidative processes in tumor cells may have a strong influence on the host response against tumors.