HIV-1-infected cells can avoid cytotoxic T lymphocyte killing by Nef-mediated down-regulation of surface MHC I. Here, we show that HIV-1 Nef inhibits MHC II restricted peptide presentation to specific T cells and thus may affect the induction of antiviral immune responses. Nef mediates this effect by reducing the surface level of mature (i.e., peptide-loaded) MHC II while increasing levels of immature MHC II, which are functionally incompetent because of their association with the invariant chain. Nef was the only HIV-1 gene product to possess this capacity, which was also observed in the context of the whole HIV-1 genome. Other proteins of the endocytic pathway were not affected by Nef expression, suggesting that Nef effects on MHC II did not result from a general alteration of the endocytic pathway. Response patterns to previously characterized mutations of Nef differed for Nef-induced modulation of mature and immature MHC II. Furthermore, the doses of Nef required to observe each of the two effects were clearly different, suggesting that Nef could affect MHC II peptide presentation through distinct mechanisms. Cooperation between those mechanisms may enable Nef to efficiently inhibit MHC II function.