Eighteen highly exposed but persistently seronegative (HEPS) women (HW) and their human immunodeficiency virus (HIV) type 1-seropositive male partners were studied for HIV-specific T cells and other host factors. Circulating HIV-specific T cells were measured by interferon-gamma enzyme-linked immunospot assays, using recombinant vaccinia virus vectors expressing HIV proteins. Nine (50%) of the HW and all HIV-seropositive persons had HIV-specific T cell responses. Only 2 (22%) of the HEPS responders recognized Env, compared with 94% of HIV-seropositive persons. A high percentage (75%) of the HW with HIV-specific T cell responses reported recent HIV exposure. Remarkably, however, long-lived HIV-specific T cells were detected in 2 HW who had an extended period (>3.9 years) of no HIV exposure. These findings have important implications for HIV vaccine design.