Our understanding of mechanisms by which the expression of IFN-gamma is regulated is limited. Herein, we identify two evolutionarily conserved noncoding sequence elements (IFNgCNS1 and IFNg CNS2) located approximately 5 kb upstream and approximately 18 kb downstream of the initiation codon of the murine Ifng gene. When linked to the murine Ifng gene (-3.4 to +5.6 kb) and transiently transfected into EL-4 cells, these elements clearly enhanced IFN-gamma expression in response to ionomycin and phorbol 12-myristate 13-acetate and weakly enhanced expression in response to T-bet. A DNase I hypersensitive site and extragenic transcripts at IFNgCNS2 correlated positively with the capacity of primary T cell subsets to produce IFN-gamma. Transcriptionally favorable histone modifications in the Ifng promoter, intronic regions, IFNgCNS2, and, although less pronounced, IFNgCNS1 increased as naïve T cells differentiated into IFN-gamma-producing effector CD8+ and T helper (TH) 1 T cells, but not into TH2 T cells. Like IFN-gamma expression, these histone modifications were T-bet-dependent in CD4+ cells, but not CD8+ T cells. These findings define two distal regulatory elements associated with T cell subset-specific IFN-gamma expression.