Human immunodeficiency virus type 1 (HIV-1)-specific cytotoxic T lymphocytes (CTL) responses provide an important defense in controlling HIV-1 replication, but they fail to control the progression of AIDS in advanced HIV-1 infection. To uncover the situation of these responses in patients with advanced HIV-1 infection, we assessed HIV-1-specific CTL responses in 20 individuals with advanced HIV-1 infection using 407 overlapping peptides spanning all expressed HIV-1 proteins using a gamma interferon-enzyme-linked immunospot (ELISpot) assay. In comparison to 20 individuals with moderately advanced HIV-1 infection, HIV-1-specific CTL responses were significantly decreased (P=0.044) and less peptides could be recognized (P=0.05) in advanced HIV-1 infection. Weakening of Env-gp120 and Gag-specific CTL responses contributed importantly to the decrease of CTL magnitude (P=0.042 and 0.078, respectively), while Env-gp41-specific CTL responses were relatively stronger during the end-stage of HIV-1 infection (P<0.001). Nef and Env-gp41 represented the most frequently targeted HIV-1 proteins in advanced HIV-1 infection. The entropy scores of peptides targeted in two groups were not significantly different. Only the breadth and magnitude of Env-gp41-specific CTL responses were positively correlated with viral loads in advanced HIV-1 infection (P=0.005 and 0.001, respectively). These findings suggest that progressive HIV-1 infection is associated with a weakening of Env-gp120- and Gag-specific CTL responses, and a simultaneous expansion of Env-gp41-specific CTL which is likely driven by high level viral replication.