AIM To develop a safe and effective DNA vaccine for inducing humoral and cellular immunological responses against hepatitis B virus surface antigen (HBsAg). METHODS BALB/c mice were inoculated with NV-HB/s, a recombinant plasmid that had been inserted S gene of hepatitis B virus genome and could express HBsAg in eukaryotes. HBsAg expression was measured by ABC immunohis-tochemical assay, generation of anti-HBs by ELISA and cytotoxic T lymphocyt e (CTL), by MTT method, existence of vaccine DNA by Southern blot hybridization and activation of oncogene C-myc by in situ hybridization. RESULTS With NV-HB/s vaccination by intramuscular injection, anti-HBs was initially positive 2 weeks after inoculation while all mice tested were HBsAg positive in the muscles. The titers and seroconversion rate of anti-HBs were steadily increasing as time went on and were dose-dependent. All the mice inoculated with 100μg NV-HB/s were anti-Bs positive one month after inoculation, the titer was 11024 or more. The∶ humora l immune response was similar induced by either intramuscular or intradermal injection. CTL activities were much stronger (45.26%) in NV-HB/s DNA immunized mice as compared with those (only 6%) in plasma-derived HBsAg vaccine immunized mice. Two months after inoculation, all muscle samples were positive by Southern-blot hybridization for NV-HB/s DNA detection, but decreased to 25% and all were undetectable by in situ hybridization after 6 months. No oncogene C-myc activation was found in the muscle of inoculation site. CONCLUSION NV-HB/s could generate humoral and cellular immunological responses against HBsAg that had been safely expressed in situ by NV-HB/s vaccination.