The efficiency of different vaccination techniques to prime in vivo major histocompatibility complex class I-restricted murine cytotoxic T-lymphocyte (CTL) precursors to hepatitis B virus small surface antigen (HBsAg) was investigated. Mice were immunized either by injection of a low dose of recombinant HBsAg protein preparations (native HBsAg particles or denatured HBsAg monomers) without adjuvants, by infection with recombinant vaccinia virus carrying an HBsAg-encoding gene, or by intramuscular transfer of plasmid DNA encoding HBsAg under appropriate promoter control. In H-2d mice, an Ld-restricted, S28-39-specific CTL response was efficiently primed by all alternative vaccination techniques tested, but the most potent priming of class I-restricted CTL to HBsAg in vivo was observed with DNA immunization. Priming of anti-HBsAg CTL in H-2b mice was not detectable after infection with a recombinant vaccinia virus or after injection with exogenous recombinant HBsAg preparations. After DNA immunization, however, both Kb- and Db-restricted CTL reactivity to HBsAg emerged in H-2b mice. Hence, nucleic acid immunization revealed class I-restricted CTL responsiveness to HBsAg in a mouse strain previously considered to be a nonresponder at the CTL level. These results demonstrate that the simple technique of nucleic acid immunization not only is extremely efficient but also reveals an extended spectrum of potentially immunogenic epitopes of protein antigens.