Addition of nitric oxide (NO) donors to mitogen-activated human immunodeficiency virus type 1 (HIV-1)-infected peripheral blood mononuclear cultures produced a significant increase in virus replication, and this effect was not associated with a change in cell proliferation. This effect was only observed with T-tropic X4 or X4R5 virus but not with R5 virus. Moreover, HIV-1 replication in mitogen-stimulated cultures was partially prevented by the specific inhibitors of the inducible nitric oxide synthase (iNOS). NO donors also enhanced HIV-1 infection of the human T-cell lines, Jurkat and MT-2. We have also observed that NO leads to an enhancement of HIV-1 replication in resting human T cells transfected with a plasmid carrying the entire HIV-1 genome and activated with phorbol ester plus ionomycin. Thus, in those cultures NO donors strongly potentiated HIV-1 replication in a dose-dependent manner, up to levels comparable to those with tumor necrosis factor alpha (TNF-alpha) stimulation. Furthermore, iNOS inhibitors decreased HIV-1 replication in HIV-1-transfected T cells to levels similar to those obtained with neutralizing anti-TNF-alpha antibodies. Moreover, HIV-1 replication induced iNOS and TNF-alpha transcription in T cells and T-cell lines. Interestingly, NO donors also stimulated long terminal repeat (LTR)-driven transcription whereas iNOS inhibitors partially blocked TNF-alpha-induced LTR transcription. Therefore, our results suggest that NO is involved in HIV-1 replication, especially that induced by TNF-alpha.