The human immunodeficiency virus (HIV) Nef protein is myristoylated and plays a critical role in AIDS pathogenesis by enhancing viral replication, survival of the virus within infected cells and by facilitating its spread in vivo. We observed that, in the promonocytic cell line U937, myristoylated exogenous Nef protein activates NF-κB and AP-1, whereas unmyristoylated exogenous Nef protein does not. Using methyl-β-cyclodextrin (MβC) treatment, we observed that the activation of NF-κB and AP-1 by exogenous Nef protein is mediated primarily via lipid rafts both in U937 cells and in primary human macrophages. In agreement with this observation, exogenous Nef protein colocalized with GM1 ganglioside, a major component of lipid rafts, in U937 cells as detected by confocal microscopy. Since tumor necrosis factor alpha (TNF α ) activates NF-κB and AP-1, we investigated the role of exogenous Nef protein in TNFα-stimulated U937 cells and primary macrophages. We observed that exogenous Nef and TNFα synergistically activate NF-κB and AP-1 in U937 cells and primary macrophages resulting in enhanced stimulation of the HIV-1 long terminal repeat (LTR), and subsequently in enhanced viral replication in both chronically infected promonocytic U1 cells and acutely HIV-1-infected primary macrophages. Both enhanced LTR stimulation and viral replication following treatment with exogenous Nef and TNFα were mediated via lipid rafts. Therefore, our results indicate that exogenous Nef protein and enhanced TNFα production detected in HIV-infected subjects could synergize to fuel the progression of the disease via lipid raft-dependent stimulation of the HIV-1 provirus present in such cellular reservoirs as mononuclear phagocytes.