We have shown previously that IFN-γ pretreatment of human epidermal cells (ECs) cultured in vitro partially reverses down-regulation of surface MHC class I by HSV infection, allowing recognition by CD8 CTLs, and that HSV immediate early (IE)/early (E) proteins are the predominant targets for CD8 CTLs. In this study of 25 subjects, CD8 CTLs recognized the HSV-2 IE infected cell protein 27 (ICP27)(expressed in autologous IFN-γ-pretreated, Vaccinia virus recombinant-infected ECs) in all subjects studied, ICP4 in 89%, and ICP0 in 11%. The main hierarchy of recognition was ICP27> ICP4. ICP27 was the dominant target in 89% of subjects but showed great individual variability in the degree of cytotoxicity. CD8 cytotoxicity specific for HSV-2 IE proteins was enhanced by 48–67% when CD8 CTLs were coincubated with the combination of monophosphoryl lipid A and QS21 adjuvants at the time of Ag presentation. These adjuvants also significantly enhanced IL-12 and IFN-γ production from nonadherent mononuclear cells stimulated by HSV-2-infected ECs. Addition of IL-12 and IFN-γ at the time of initial Ag presentation enhanced CD8 cytotoxicity to levels comparable with those stimulated by the adjuvants. Addition of neutralizing Abs to IL-12 or IFN-γ inhibited CD8 T cell cytotoxicity up to 95% when a combination of the Abs were added at the time of initial Ag presentation. Therefore, the mechanism for the enhancement of CD8 T cell cytotoxicity by adjuvants in this system appears to be via increased levels of IL-12 and IFN-γ.