HIV-1 Nef is a critical determinant of pathogenicity in humans and transgenic (Tg) mice. To gain a better understanding of the molecular mechanisms by which Nef induces an AIDS-like disease in Tg mice, a mutational analysis of the N-terminal domain, involved in anchoring Nef to the plasma membrane, was carried out. The pathogenic effects of these Nef mutant alleles were evaluated in Tg mice by FACS analysis and by histopathological assessment. Mutation of the myristoylation site (G2A) completely abrogated the development of the AIDS-like organ disease in Tg mice, although partial downregulation of the CD4 cell surface protein and depletion of peripheral CD4+ T-cells, but not of CD4(+)CD8+ thymocytes, still occurred. Despite that, the peripheral CD4+ T cells expressing Nef(G2A) show normal spontaneous proliferation in vivo or after stimulation in vitro, including in an allogenic mixed leukocyte reaction (MLR). Three other internal deletion mutants of Nef, spanning amino acids 8-17 (Nef(Delta8-17)), 25-35 (Nef(Delta25-35)), and 57-66 (Nef(Delta57-66)), were also studied. Nef(Delta8-17) retained full pathogenic potential, although Nef(Delta25-35) and Nef(Delta57-66) Tg mice were free of organ disease. However, Nef(Delta25-35) Tg mice exhibited disorganization of thymic architecture and a partial depletion of peripheral CD4+ T cells. These data indicate that myristoylation and other regions at the N-terminus of Nef (aa 25-35 and 57-66) are involved in mediating severe T-cell phenotypes and organ disease, although residues 8-17 are dispensable for these Nef functions. In addition, these results indicate that at least some of the CD4+ T-cell phenotypes can develop independently of the other AIDS-like organ phenotypes. This apparent segregation of different Nef-mediated phenotypes suggests distinct mechanisms of Nef action in different populations of target cells, and may be relevant to human AIDS.