NF-kappaB/IkappaB proteins play a major role in the transcriptional regulation of human immunodeficiency virus, type-1 (HIV-1). In the case of simian immunodeficiency virus (SIV) the cellular factors required for the viral transcriptional activation and replication in vivo remain undefined. Here, we demonstrate that the p50/p65 NF-kappaB transcription factors enhanced the Tat-mediated transcriptional activation of SIVmac239. In addition, IkappaB-alpha S32/36A, a proteolysis-resistant inhibitor of NF-kappaB, strongly inhibited the Tat-mediated transactivation of SIVmac239. Based on this evidence, we have generated a self-regulatory virus by endowing the genome of SIV-mac239 with IkappaB-alpha S32/36A; the resulting virus, SIVIkappaB-alpha S32/36A, was nef-deleted and expressed the NF-kappaB inhibitor. We show that SIVIkappaB-alpha S32/36A was highly and stably attenuated both in cell cultures and in vivo in rhesus macaque as compared with a nef-deleted control virus. Moreover, the high attenuation was associated with a robust immune response as measured by SIV-specific antibody production, tetramer, and intracellular IFN-gamma staining of SIV gag-specific T cells. These results underscore the crucial role of NF-kappaB/IkappaB proteins in the regulation of SIV replication both in cell cultures and in monkeys. Thus, inhibitors of NF-kappaB could efficiently counteract the SIV/HIV replication in vivo and may assist in developing novel approaches for AIDS vaccine and therapy.