In 2003, we described a small cohort of subjects (n= 6) who possessed no detectable serum Abs to HSV-1 or HSV-2 and no clinical or virological evidence of mucosal HSV infection yet possessed consistently detectable HSV-specific T cell responses measured primarily by lymphoproliferative (LP) and CTL assays to whole HSV-2 Ag. We termed these persons immune seronegative (IS). This report characterizes the T cell responses in 22 IS subjects largely recruited from studies of HSV-seronegative subjects in ongoing sexual relationships with HSV-2–seropositive (HSV-2+) partners using pools of overlapping peptides spanning 16 immuno-prevalent HSV-2 proteins. Overall, 77% of IS subjects had HSV-specific LP responses, 85% had IFN-γ ELISPOT responses to at least one HSV-2 peptide pool, and 55% had both LP and IFN-γ ELISPOT responses. In some cases, IFN-γ ELISPOT responses were in excess of 500 spot-forming cells per 10 6 PBMCs and persisted for over 5 y. Although HSV-2+ subjects (n= 40) had frequent responses to glycoproteins and tegument and immediate-early (IE) proteins of HSV-2, T cell responses in IS subjects were directed primarily at UL39 and the IE proteins ICP4 and ICP0. These data suggest that the antigenic repertoire of T cells in IS subjects is skewed compared with that of HSV-2+ subjects and that IS subjects had more frequent T cell responses to IE proteins and infrequent T cell responses to virion components. Understanding the mechanism (s) by which such responses are elicited may provide important insights in developing novel strategies for preventing acquisition of sexually acquired HSV-2.