The cell surface expression of the envelope glycoproteins (Envs) of primate immunodeficiency viruses is, at least in part, regulated by endocytosis signal(s) located in the Env cytoplasmic domain. Here, we show that a membrane proximal signal that directs the simian immunodeficiency virus (SIV) Env to clathrin-coated pits, and is conserved in all SIV and human immunodeficiency virus Envs, conforms to a YxxØ motif (where x can be any amino acid and Ø represents a large hydrophobic residue). This motif is similar to that described for a number of cellular membrane proteins. By surface plasmon resonance we detected a high affinity interaction between peptides containing this membrane proximal signal and both AP1 and AP2 clathrin adaptor complexes. Mutation of the tyrosine in this membrane proximal motif in a SIV Env with a prematurely truncated cytoplasmic domain leads to a > or = 25-fold increase in Env expression on infected cells. By contrast, the same mutation in an Env with a full-length cytoplasmic domain increases cell surface expression only 4-fold. We show that this effect results from the presence of additional endocytosis signals in the full-length cytoplasmic domain. Chimeras containing CD4 ecto- and membrane spanning domains and a full-length SIV Env cytoplasmic domain showed rapid endocytosis even when the membrane proximal tyrosine-based signal was disrupted. Mapping experiments indicated that at least some of the additional endocytosis information is located between residues 743 and 812 of Env from the SIVmac239 molecular clone. Together, our findings indicate that the cytoplasmic domain of SIV Env contains multiple endocytosis and/or trafficking signals that modulate its surface expression on infected cells, and suggest an important role for this function in pathogenesis.