Human immunodeficiency virus (HIV-1) utilizes the NF-kappaB/Rel proteins to regulate transcription through NF-kappaB binding sites in the HIV-1 long terminal repeat (LTR). Normally, NF-kappaB is retained in the cytoplasm by inhibitory IkappaB proteins; after stimulation by multiple activators including viruses, IkappaBalpha is phosphorylated and degraded, resulting in NF-kappaB release. In the present study, we examined the effect of tetracycline-inducible expression of transdominant repressors of IkappaBalpha (TD-IkappaBalpha) on HIV-1 multiplication using stably selected Jurkat T cells. TD-IkappaBalpha was inducibly expressed as early as 3 h after doxycycline addition and dramatically reduced both NF-kappaB DNA binding activity and LTR-directed gene activity. Interestingly, induced TD-IkappaBalpha expression also decreased endogenous IkappaBalpha expression to undetectable levels by 24 h after induction, demonstrating that TD-IkappaBalpha repressed endogenous NF-kappaB-dependent gene transcription. TD-IkappaBalpha expression also sensitized Jurkat cells to tumor necrosis factor-induced apoptosis. De novo HIV-1 infection of Jurkat cells was dramatically altered by TD-IkappaBalpha induction, resulting in inhibition of HIV-1 multiplication, as measured by p24 antigen, reverse transcriptase, and viral RNA. Given the multiple functions of the NF-kappaB/IkappaB pathway, TD-IkappaBalpha expression may interfere with HIV-1 multiplication at several levels: LTR-mediated transcription, Rev-mediated export of viral RNA, inhibition of HIV-1-induced pro-inflammatory cytokines, and increased sensitivity of HIV-1-infected cells to apoptosis.