In asymptomatic carriers, the vast majority of human immunodeficiency virus type 1 (HIV-1) infection is non-productive whilst the clinical stage of disease is associated with significant virus expression. Virus-specific CD8+ T-cell functions are believed to play a major role in the generation of heterogeneous virus populations and in subsequent disease progression. Here, we prepared two types of HIV-1 isolate by culturing whole and CD8+ T cell-depleted peripheral blood mononuclear cells (PBMCs) from five asymptomatic carriers. The former is expected to be escape variant populations, whereas the latter would be mixed populations including the former viruses. The analyses of Nef and Env V3 sequence variations of viruses in a total of 77 and 44 DNA clones, respectively, allowed a direct comparison to be made of the differences between the paired isolates. Comparison of Nef sequences between the paired isolates showed them to be more distinct in two carriers with a relatively stable CD4/CD8 ratio (Nos 68 and 69), than in two other carriers with similar CD4/CD8 ratios (Nos 53 and 57), or in carrier No. 67, which had an extremely lower CD4/CD8 ratio. By contrast, a distinction between the paired isolates by use of the Env V3 sequences was only apparent in the latter three carriers. These results indicate that the predominant populations of HIV-1 in Nos 68 and 69 were sensitive to selective pressure from Nef-specific CD8+ T-cells, while those in Nos 53, 57, and 67 were sensitive to pressure from V3-specific CD8+ T-cells. It is noteworthy that Nos 53 and 57 progressed to an AIDS-related complex shortly and several years after this examination. These data suggest that HIV-1-induced pathogenesis is more strongly associated with the generation of variant nef alleles than with env V3 variants, and that these arise by CD8+ T-cell pressure.