Understanding how highly HIV-exposed individuals remain HIV uninfected may be useful for HIV vaccine design and development of new HIV prevention strategies. To elucidate mechanisms associated with resistance to HIV infection, immunologic and genetic factors were examined in 14 HIV-exposed but persistently seronegative (HEPS) female sex workers from Chiang Rai, northern Thailand and in ethnically matched, HIV-positive (n = 9) and HIV-negative women (n = 9). The HEPS women were identified in a study of commercial sex workers who had an HIV-1 incidence of 20.3 per 100 person-years. A high frequency of HLA-A11 was observed in HEPS women (86%) compared with northern Thai controls (56%). HIV-specific cytotoxic T lymphocyte (CTL) lytic responses were detected in cryopreserved peripheral blood mononuclear cells (PBMCs), using HLA-A-matched subtype E HIV-1 peptides in four of seven (57%) HEPS women, eight of eight HIV-positive women, and zero of nine HIV-negative unexposed controls (p = 0.019 HEPS women vs. HIV-negative controls). CTL lysis levels were low, but responses were detected to peptides from Nef, Pol, Gag, and Env. Nef responses predominated in HEPS women. Compared with controls, HEPS women tended to have higher frequencies of CCR5 promotor 59402GG and SDF-1 3'UTR 801A genotypes known to influence HIV transmission or course of disease. HEPS women also had higher levels of spontaneous RANTES production by PBMCs than other groups. Each of these factors could potentially contribute to HIV resistance. As most HEPS women had one or more of these factors, they may prevent HIV infection synergistically by blocking HIV cell entry, delaying its dissemination, or killing HIV-infected cells.