Live-attenuated human immunodeficiency viruses (HIVs) are candidates for Acquired Immunodeficiency Syndrome (AIDS) vaccine. Based on the simian immunodeficiency virus (SIV) model for AIDS, loss-of-function (e.g. deletion of accessory genes such as nef) has been forwarded as a primary approach for creating enfeebled, but replication-competent, HIV-1/SIV. Regrettably, recent evidence suggests that loss-of-function alone is not always sufficient to prevent the emergence of virulent mutants. New strategies that attenuate via mechanisms distinct from loss-of-function are needed for enhancing the safety phenotype of viral genome. Here, we propose gain-of-function to be used simultaneously with loss-of-function as a novel approach for attenuating HIV-1. We have constructed an HIV-1 genome carrying the cDNA of a proteolysis-resistant nuclear factor-kappaB inhibitor (IkappaB-alphaS32/36A) in the nef region. HIV-1 expressing IkappaB-alphaS32/36A down-regulates viral expression and is highly attenuated in both Jurkat and peripheral blood mononuclear cells. We provide formal proof that the phenotypic and attenuating characteristics of IkappaB-alphaS32/36A permit its stable maintenance in a live, replicating HIV-1 despite 180 days of forced ex vivo passaging in tissue culture. As compared with other open-reading frames embedded into HIV/SIV genome, this degree of stability is unprecedented. Thus, IkappaB-alphaS32/36A offers proof-of-principle that artifactually gained functions, when used to attenuate the replication of live HIV-1, can be stable. These findings illustrate gain-of-function as a feasible strategy for developing safer live-attenuated HIVs to be tested as candidates for AIDS vaccine.