SIV and HIV Nef proteins disrupt T‐cell receptor machinery by down‐modulating cell surface expression of CD4 and expression or signaling of CD3‐TCR. Nef also down‐modulates class I major histocompatibility complex (MHC) surface expression. We show that SIV and HIV‐1 Nefs down‐modulate CD28, a major co‐stimulatory receptor that mediates effective T‐cell activation, by accelerating CD28 endocytosis. The effects of Nef on CD28, CD4, CD3 and class I MHC expression are all genetically separable, indicating that all are selected independently. In cells expressing a Nef‐green fluorescent protein (GFP) fusion, CD28 co‐localizes with the AP‐2 clathrin adaptor and Nef‐GFP. Mutations that disrupt Nef interaction with AP‐2 disrupt CD28 down‐regulation. Furthermore, HIV and SIV Nefs use overlapping but distinct target sites in the membrane‐proximal region of the CD28 cytoplasmic domain. Thus, Nef probably induces CD28 endocytosis via the AP‐2 pathway, and this involves a ternary complex containing Nef, AP‐2 and CD28. The likely consequence of the concerted down‐regulation of CD28, CD4 and/or CD3 by Nef is disruption of antigen‐specific signaling machineries in infected T cells following a productive antigen recognition event.