The long terminal repeat of human immunodeficiency virus, type 1 (HIV-1) contains an NF-kappaB enhancer and is potently inhibited by IkappaB-alphaS32/36A, a proteolysis-resistant inhibitor of NF-kappaB transacting factors. The evidence that NF-kappaB is dispensable for HIV-1 expression raises the question of whether IkappaB-alpha represses the HIV-1 transcription by mechanisms distinct from NF-kappaB inhibition. Here, we report that IkappaB-alpha negatively regulates the HIV-1 expression and replication in an NF-kappaB-independent manner by directly binding to Tat, which results in the nuclear export and cytoplasmic sequestration of the viral transactivator. The sequence of IkappaB-alpha required for Tat inhibition spans from amino acids 72 to 287 and includes the nuclear localization signal, the carboxyl-terminal nuclear export signal, and the binding site for the arginine-rich domain of Tat. This novel mechanism of cross-talk between Tat and IkappaB-alpha provides further insights into the mechanisms of HIV-1 regulation and could assist in the development of novel strategies for AIDS therapy.