The maximal virulence of HIV-1 requires Nef, a virally encoded peripheral membrane protein. Nef binds to the adaptor protein (AP) complexes of coated vesicles, inducing an expansion of the endosomal compartment and altering the surface expression of cellular proteins including CD4 and class I major histocompatibility complex. Here, we show that Nef stabilizes the association of AP-1 and AP-3 with membranes. These complexes remained with Nef on juxtanuclear membranes despite the treatment of cells with brefeldin A, which induced the release of ADP-ribosylation factor 1 (ARF1) from these membranes to the cytosol. Nef also induced a persistent association of AP-1 and AP-3 with membranes despite the expression of dominant-negative ARF1 or the overexpression of an ARF1-GTPase activating protein. Mutational analysis indicated that the direct binding of Nef to the AP complexes is essential for this stabilization. The leucine residues of the EXXXLL motif found in Nef were required for binding to AP-1 and AP-3 in vitro and for the stabilization of these complexes on membranes in vivo, whereas the glutamic acid residue of this motif was required specifically for the binding and stabilization of AP-3. These data indicate that Nef mediates the persistent attachment of AP-1 and AP-3 to membranes by an ARF1-independent mechanism. The stabilization of these complexes on membranes may underlie the pleiotropic effects of Nef on protein trafficking within the endosomal system.