Among the pleiotropic effects of Nef proteins of HIV and SIV, down-modulation of cell surface expression of CD4 is a prominent phenotype. It has been presumed that Nef proteins accelerate endocytosis of CD4 by linking the receptor to the AP-2 clathrin adaptor. However, the related AP-1 and AP-3 adaptors have also been shown to interact with Nef, hinting role(s) for these complexes in intracellular retention of CD4. By using genetic inhibitors of endocytosis and siRNA induced knock down of AP-2, we show that accelerated CD4 endocytosis is not a dominant mechanism of HIV-1 (NL4-3 strain) Nef in epithelial cells, T lymphocyte cell lines or peripheral blood lymphocytes (PBLs). Furthermore, we show that both the CD4 recycling from the plasma membrane and the nascent CD4 in transit to the plasma membrane are susceptible to intracellular retention in HIV-1 Nef expressing cells. In contrast, AP-2 mediated enhanced endocytosis constitutes the predominant mechanism for SIV (MAC-239 strain) Nef induced downregulation of human CD4 in human cells.