Nef, a approximately 200 residue multifunctional regulatory protein of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), interacts with components of host cell signal transduction and clathrin-dependent protein sorting pathways. The downregulation of surface CD4 molecules and major histocompatibility complex (MHC) class I antigens by Nef is believed to be important in AIDS pathogenesis [1-7]. Nef contains a globular core domain and two disordered segments--a myristylated arm at the amino terminus and a carboxy-terminal loop projecting from the globular core [8,9]. Here, we aimed to determine the sorting signals in HIV-1 Nef that were responsible for its involvement in the clathrin-mediated pathway. We found that a sequence in the carboxy-terminal disordered loop of Nef is essential for downregulation of CD4. This sequence resembles the dileucine motif, one of two well-characterized sorting signals that target membrane proteins to clathrin-coated vesicles. The dileucine-motif-containing segment of Nef bound directly and specifically to the beta-adaptin subunit of the clathrin adaptor complexes AP-1 and AP-2, which are responsible for recruiting sorted proteins into coated pits. Unlike wild-type Nef, a mutant form of Nef that lacked the dileucine motif did not localize to clathrin-coated pits and did not downregulate CD4 expression, although it could downregulate MHC class I surface expression. Thus, the dileucine motif in HIV-1 is required for CD4 downregulation and for interaction with clathrin adaptor complexes.