The human immunodeficiency virus (HIV) nef gene encodes a 27 kDa myristoylated cytosolic protein that has an important role in the pathogenesis of AIDS. One function of Nef is the down-regulation of CD4 and MHC class I surface molecules in HIV-infected cells. Nef directly isolated from an infected individual (KS2), who could be defined as a long-term non-progressor, was compared with Nef from a standard laboratory strain, HIV-1 NL4-3. KS2 Nef protein was characterized by its lowered ability to down-regulate CD4, while still maintaining the ability to down-regulate MHC class I. The ability of KS2 Nef to down-regulate CD4 was more prominent when CD4 was measured 2-3 days after transfer of the nef gene to the target cells, and also when the effect was measured in CD4(+)-enriched primary T cells. The amino acid sequence analysis indicated that the most notable feature of KS2 Nef was lack of the two glutamic acids: the EE(155) region. When the EE(155) region was added to KS2 Nef, the CD4 down-regulation ability was increased almost to the level of NL4-3 Nef. Conversely, when the EE(155) region was deleted from NL4-3, its CD4 down-regulation ability was dramatically impaired. These data suggested that the EE(155) region plays an important role(s) in the down-regulation of CD4 by Nef protein and also that primary nef sequences could be very useful in identifying the original biological functions of Nef in vivo.