IFN-γ production is dramatically reduced in T cells from mice bearing large mammary tumors. This inhibition of IFN-γ gene expression occurs mostly in CD4+ T cells, as determined by ELISA and reverse transcriptase-PCR. The effects of known mammary tumor factors in normal T cells and its subsets were evaluated. Pretreatment with granulocyte-macrophage CSF resulted in increased IFN-γ levels by T cells, while PGE 2 pretreatment equally decreased the levels of this cytokine in CD4+ and CD8+ T cells from normal mice. Interestingly, phosphatidyl serine (PS) down-regulated the IFN-γ production of CD4+, but not that of CD8+, T cells. Methylation analysis indicated that the CpG dinucleotide in SnaBI site of the IFN-γ 5′ promoter flank region was hypermethylated in CD4+, but not in CD8+, T cells of large tumor bearers and of normal mice pretreated with PS. Electrophoresis mobility shift assay using an oligonucleotide probe corresponding to the IFN-γ promoter core region sequence showed a greatly reduced binding of a 90-kDa nuclear protein in CD4+ T cells from tumor bearers and in those from PS-pretreated normal mice. Since IL-2 production is not affected in either CD4+ or CD8+ T cells from tumor bearers, these studies indicate that IFN-γ production can be regulated independently from that of other type 1 cytokines in vivo. Our data further suggest that PS is involved in IFN-γ gene down-regulation during mammary tumorigenesis and contributes to the generalized immunosuppression associated with tumor growth.