Nef is a myristoylated protein of 27 to 35 kDa that is conserved in primate lentiviruses. In vivo, Nef is required for high viral load and full pathological effects. In vitro, Nef has at least four activities: induction of CD4 and major histocompatibility complex (MHC) class I downregulation, enhancement of viral infectivity, and alteration of T-cell activation pathways. We previously reported that the Nef protein from human immunodeficiency virus type 1 interacts with a novel human thioesterase (hTE). In the present study, by mutational analysis, we identified a region of the Nef core, extending from the residues D108 to W124, that is involved both in Nef-hTE interaction and in Nef-induced CD4 downregulation. This region of Nef is located on the oligomer interface and is in close proximity to the putative CD4 binding site. One of the mutants carrying a mutation in this region, targeted to the conserved residue D123, was also found to be defective in two other functions of Nef, MHC class I downmodulation and enhancement of viral infectivity. Furthermore, mutation of this residue affected the ability of Nef to form dimers, suggesting that the oligomerization of Nef may be critical for its multiple functions.