Persistent infections caused by such agents as the human immunodeficiency virus, hepatitis B virus, Epstein–Barr virus, etc., present formidable medical problems. A defining characteristic of these infections is that anti-viral cytotoxic T lymphocytes (CTL) may be lost or, if present, fail to clear the infection. Here we report a vaccination strategy which was successful in generating lytic CTL in persistently infected mice. Vaccination with an immunodominant CTL epitope derived from the nucleoprotein of lymphocytic choriomeningitis virus (LCMV) delivered in the form of a lipopeptide incorporating a universal CD4 helper epitope successfully induced lytic MHC-restricted CTL in mice persistently infected with LCMV since birth. However, induction of such CTL did not eliminate the virus, most likely because the CTL were generated at low frequencies and had 2 to 3 logs lower affinity than CTL generated in uninfected mice inoculated with the vaccine. Both CTL populations from either uninfected or persistently infected mice produced significant and similar amounts of interferon-γ and IL-6. Vaccine-induced low-affinity CTL were still inadequate at complete removal of the virus when combined with LCMV-specific CD4 helper T lymphocytes. Thus, our results establish that CTL can be generated in persistently infected mice and that a crucial factor for clearing viral infection is the affinity of the CTL.