CD8+ cytotoxic T lymphocytes (CTL) recognize antigens (Ag) associated with class I major histocompatibility complex (MHC) molecules. Endogenously synthesized protein Ag are processed into peptides in the cytoplasm and transported to the endoplasmic reticulum where they are bound by class I proteins. Exogenous Ag do not enter the class I processing pathway of most cells and thus do not activate CD8+ CTL. Nevertheless, several investigators have reported that immunization with exogenous Ag can activate CD8+ T cells that have immunoregulatory activity. To determine how exogenous Ag entered the class I pathway in vivo and whether immunosuppressive CD8+ T cells were cytolytic, we have shown in this report that injection with OVA emulsified in the complete Freund's adjuvant (CFA) primed CD8+, class I MHC-restricted, OVA-specific CTL in mice. These CTL recognize the OVA257-264 epitope, produce tumor necrosis factor-alpha and interferon-gamma upon activation. Both oil and mycobacteria components in CFA were required for inducing CTL responses. Priming was not attributed to direct sensitization of class I-bearing cells by contaminating peptides. Rather, phagocytic cells, but not CD4+ helper T cells, were required for priming CD8+ CTL by OVA-CFA. Thus, OVA in CFA is taken up by antigen-presenting cells and processed into the class I pathway by phagocytic cells in vivo. In addition, CTL induced by OVA-CFA suppressed the antibody response to OVA in adoptive recipients. These results suggest that CD8+ CTL specific for exogenous proteins might be routinely stimulated by injecting proteins in conventional adjuvants and that such cells have the potential to regulate immune responses in vivo.