Tristetraprolin (TTP) regulates expression at the level of mRNA decay of several cytokines, including the T cell-specific cytokine, interleukin-2. We performed experiments to determine whether another T cell-specific cytokine, interferon-gamma (IFN-gamma), is also regulated by TTP and found that T cell receptor-activated T cells from TTP knock-out mice overproduced IFN-gamma mRNA and protein compared with activated T cells from wild-type mice. The half-life of IFN-gamma mRNA was 23 min in anti-CD3-stimulated T cells from wild-type mice, whereas it was 51 min in anti-CD3-stimulated T cells from TTP knock-out mice, suggesting that the overexpression of IFN-gamma mRNA in TTP knock-out mice was due to stabilization of IFN-gamma mRNA. Insertion of a 70-nucleotide AU-rich sequence from the murine IFN-gamma 3'-untranslated region, which contained a high affinity binding site for TTP, into the 3'-untranslated region of a beta-globin reporter transcript conferred TTP-dependent destabilization on the beta-globin transcript. Together these results suggest that TTP binds to a functional AU-rich element in the 3'-untranslated region of IFN-gamma mRNA and mediates rapid degradation of the IFN-gamma transcript. Thus, TTP plays an important role in turning off IFN-gamma expression at the appropriate time during an immune response.