The envelope glycoprotein (Env) of human immunodeficiency virus, type 1 (HIV-1) undergoes rapid internalization after its transport to the cell surface. Env internalization is dependent upon information contained within the cytosolic domain of the protein. Here, we report that the cytosolic domain of Env binds specifically to the medium chain, mu 2, of the clathrin-associated protein complex AP-2, as well as to the complete AP-2 complex. The Env cytosolic domain contains two highly conserved tyrosine-based motifs (Y712SPL and Y768HRL), both of which are capable of binding to mu 2 when presented as short peptides. However, only the membrane-proximal motif Y712SPL binds to mu 2 and is required for internalization in the context of the whole cytosolic domain of Env. A glycine residue (Gly711) adjacent to the Y712SPL motif is also important for binding to mu 2/AP-2 and internalization. These observations suggest that the accessibility of the membrane-proximal GY712SPL to mu 2/AP-2 determines its function as a signal for recruitment of HIV-1 Env into clathrin-coated pits and its ensuing internalization.