The Nef protein of the human immunodeficiency virus type 1 (HIV-1) plays a crucial role in AIDS pathogenesis by modifying host cell signaling pathways. We investigated the effects of Nef on the NADPH oxidase complex, a key enzyme involved in the generation of reactive oxygen species during the respiratory burst in human monocyte/macrophages. We have recently shown that the inducible expression of HIV-1 Nef in human macrophages cell line modulates in bi-phasic mode the superoxide anion release by NADPH oxidase, inducing a fast increase of the superoxide production, followed by a delayed strong inhibition mediated by Nef-induced soluble factor(s). Our study is focused on the molecular mechanisms involved in Nef-mediated activation of NADPH oxidase and superoxide anion release. Using U937 cells stably transfected with different Nef alleles, we found that both Nef membrane localization and intact SH3-binding domain are needed to induce superoxide release. The lack of effect during treatment with a specific MAPK pathway inhibitor, PD98059, demonstrated that Nef-induced superoxide release is independent of Erk1/2 phosphorylation. Furthermore, Nef induced the phosphorylation and then the translocation of the cytosolic subunit of NADPH oxidase complex p47(phox) to the plasma membrane. Adding the inhibitor PP2 prevented this process, evidencing the involvement of the Src family kinases on Nef-mediated NADPH oxidase activation. In addition, LY294002, a specific inhibitor of phosphoinositide 3-kinase (PI3K) inhibited both the Nef-induced p47(phox) phosphorylation and the superoxide anion release. These data indicate that Nef regulates the NADPH oxidase activity through the activation of the Src kinases and PI3K.