A variety of simian immunodeficiency virus (SIVmac) nef mutants have been investigated to clarify which in vitro Nef functions contribute to efficient viral replication and pathogenicity in rhesus macaques. Most of these nef alleles, however, were only functionally characterized for their ability to down-modulate CD4 and class I major histocompatibility complex (MHC-I) cell surface expression and to enhance SIV replication and infectivity. To obtain information on the in vivo relevance of more recently established Nef functions, we examined the ability of a large panel of constructed SIVmac Nef mutants and of variants that emerged in infected macaques to down-regulate CD3, CD28, and MHC-II and to up-regulate the MHC-II-associated invariant chain (Ii). We found that all these four Nef functions were restored in SIV-infected macaques. In most cases, however, the initial mutations and the changes selected in vivo affected several in vitro Nef functions. For example, truncated Nef proteins that emerged in animals infected with SIVmac239 containing a 152-bp deletion in nef efficiently modulated both CD3 and Ii surface expression. Overall, our results suggest that the effect of Nef on each of the six cellular receptors investigated contributes to viral fitness in the infected host but also indicate that modulation of CD3, MHC-I, MHC-II, or Ii surface expression alone is insufficient for SIV virulence.