Background Studies of highly exposed persistently seronegative (HEPS) individuals may provide valuable information on mechanisms of protection and on vaccine design. Cellular immune responses play a critical role in containing human immunodeficiency virus. However, the cellular immune responses in HEPS individuals have not been thoroughly assessed at the entire viral genome level. Methods Ten HEPS Chinese with a history of frequent penetrative vaginal intercourse (mean frequency, at least once a week), with some unprotected sexual contact occurring in the weeks or days immediately before enrollment, 25 HIV-1 seropositive individuals, 10 HIV-1-seronegative healthy individuals with low-risk sexual behavior and no history suggestive of exposure to HIV-1 infection were enrolled. HIV-1-specific T cell responses were comprehensively analyzed by an interferon-γ Elispot assay against 770 overlapping peptides spanning all HIV-1 proteins. Results HIV-1-specific T-cell responses of interferon-γ secretion were identified in 3 (30%) out of 10 HEPS individuals; the specific cytotoxic T lymphocytes were targeted at Pol (2/10), Env (2/10), and Tat (1/10). HIV-1-specific T-cell responses of interferon-γ secretion were identified in 20 (80%) out of 25 seropositive intravenous drug users (IDUs), revealing that all HIV-1 proteins and protein subunits could serve as targets for HIV-1-specific CD8+ T cell responses with 85% recognizing Gag, 80% recognizing Nef, 75% recognizing Pol, 60% recognizing Env, 55% recognizing Vpu, 45% recognizing Vpr, 20% recognizing Vif, 20% recognizing Tat and 15% recognizing Rev in these seropositive individuals. None of the seronegative healthy individuals gave the positive T-cell responses. Conclusions About 30% of HEPS Chinese mounted HIV-1 specific T cell immune responses. Cell-mediated immunity against HIV-1 may be developed through non-productive infections. Background Studies of highly exposed persistently seronegative (HEPS) individuals may provide valuable information on mechanisms of protection and on vaccine design. Cellular immune responses play a critical role in containing human immunodeficiency virus. However, the cellular immune responses in HEPS individuals have not been thoroughly Methods at the entire viral genome level. Methods Ten HEPS Chinese with a history of frequently penetrative vaginal intercourse (mean frequency, at least once a week), with some unprotected sexual contact occurring in the weeks or days immediately before enrollment, 25 HIV-1 seropositive individuals, 10 HIV-1-seronegative healthy individuals with low-risk sexual behavior and no history suggestive of exposure to HIV-1 infection were enrolled. HIV-1-specific T cell responses were analyzed by an interferon-γ Elispot assay against 770 overlapping peptides spanning all HIV-1 proteins. Results HIV-1-specific T-cell responses of interferon-γ The specific cytotoxic T lymphocytes were targeted at Pol (2/10), Env (2/10), and Tat (1/10). HIV-1-specific T-cell responses of interferon-γ secretion were identified in 20 (80%) out of 25 seropositive intravenous drug users (IDUs), revealing that all HIV-1 proteins and protein subunits could serve serve targets for HIV-1-specific CD8 + T cell responses with 85% recognizing Gag, 80% recognizing Nef, 75% recognizing Pol, 60% recognizing Env, 55% recognizing Vpu, 45% recognizing Vpr, 20% recognizing Vif, 20% recognizing Tat and 15% recognizing Rev in these seropositive None of the seronegative healthy individuals gave the positive T-cell responses. Conclusions About 30% of HEPS Chinese mounted HIV-1 specific T cell immune responses. Cell-mediated immunity against HIV-1 may be developed through non-productive infections.