We investigated the stimulation of a MHC class I-restricted response of CTL to the SV40 large tumor Ag (T-Ag) by different vaccination strategies in H-2d and H-2b mice. Immunization with plasmid DNA or exogenous T-Ag, or infection with SV40, primed CTL to T-Ag in H-2b mice; these three different types of Ag delivery primed T-Ag-specific CTL populations with similar epitope/restriction specificities. In H-2d mice, i.m. immunization with plasmid DNA, but neither immunization with exogenous protein Ag nor SV40 infection, primed CTL to T-Ag. T-Ag-specific H-2d CTL were primed by DNA-based immunization in vivo, expressed the CD4-CD8+ phenotype, and were L(d)-restricted. In H-2d (DBA/2) mice, T-Ag-specific immune responses primed by plasmid DNA injection, but not those primed by exogenous T-Ag or SV40 infection, mediated CD8+ CTL-dependent rejection of T-Ag-expressing P815/T tumor grafts. The data indicate that immunization by plasmid DNA injection is an efficient strategy to induce class I-restricted CTL responses against oncogene-encoded Ags of low immunogenicity that mediate tumor rejection.