Triggering of the TCR by cognate peptide/MHC ligands induces expression of IkappaBNS, a member of the IkappaB family of NF-kappaB inhibitors whose expression is associated with apoptosis of immature thymocytes. To understand the role of IkappaBNS in TCR triggering, we created a targeted disruption of the IkappaBNS gene. Surprisingly, mice lacking IkappaBNS show normal thymic progression but both thymocytes and T cells manifest reduced TCR-stimulated proliferation. Moreover, IkappaBNS knockout thymocytes and T cells produce significantly less IL-2 and IFN-gamma than wild-type cells. Transfection analysis demonstrates that IkappaBNS and c-Rel individually increase IL-2 promoter activity. The effect of IkappaBNS on the IL-2 promoter, unlike c-Rel, is dependent on the NF-kappaB rather than the CD28RE site; mutation of the NF-kappaB site extinguishes the induction of transcription by IkappaBNS in transfectants and prevents association of IkappaBNS with IL-2 promoter DNA. Microarray analyses confirm the reduction in IL-2 production and some IFN-gamma-linked transcripts in IkappaBNS knockout T cells. Collectively, our findings demonstrate that IkappaBNS regulates production of IL-2 and other cytokines induced via "strong" TCR ligation.