Induction of tumor-specific immune responses results in the inhibition of tumor development. However, tumors recur because of the tumor immunoediting process that facilitates development of escape mechanisms in tumors. It is not known whether tumor escape is an active process whereby anti-tumor immune responses induce loss or downregulation of the target antigen in the antigen-positive clones. To address this question, we used rat neu-overexpressing mouse mammary carcinoma (MMC) and its relapsed neu antigen-negative variant (ANV). ANV emerged from MMC under pressure from neu-specific T cell responses in vivo. We then cloned residual neu antigen-negative cells from MMC and residual neu antigen-positive cells from ANV. We found marked differences between these neu-negative clones and ANV, demonstrating that the residual neu-negative clones are probably not the origin of ANV. Since initial rejection of MMC was associated with the presence of IFN-gamma-secreting T cells, we treated MMC with IFN-gamma and showed that IFN-gamma could induce downregulation of neu expression in MMC. This appears to be due to methylation of the neu promoter. Together, these data suggest that neu antigen loss is an active process that occurs in primary tumors due to the neu-targeted anti-tumor immune responses.