Nef is one of six HIV-1 accessory proteins and directly contributes to AIDS progression. Nef associates with membranes and may require a transition from a solution conformation to a membrane-associated conformation. It has been hypothesized that a transition from a “closed” conformational form to an “open” form enables interaction of Nef with cellular proteins. Despite its obvious disease importance, there is little or no direct information about the conformation of membrane-bound Nef. In this work we used neutron reflection to reveal details of the conformation of membrane-bound Nef. Nef was bound through an N-terminal His tag to Langmuir monolayers of DPPC mixed with a synthetic metal-chelating lipid. Several methods were found to achieve a dense monomolecular layer of membrane-bound Nef, despite its tendency to form oligomers at high concentration. At the conditions of this initial study (65% DSIDA 35 mol% DPPC, 20 mM Tris, 20 C, pH 8.2), for the large majority of the bound population the core domain of membrane-bound Nef lies within a few A of the lipid headgroups. The N-terminal arm is directly against the lipid headgroups with a small portion inserted. The results also indicate that the disordered loop extends from the core domain into the solution. The data also suggest that for a very small fraction of the bound population the N-terminal arm extends normal to the membrane and the core domain is displaced ∼50 A from the membrane. Some ramifications of these results for the activity of Nef are discussed.