Hepatitis B surface antigen (HBsAg) complexed with anti-HBs is more immunogenic than HBsAg alone in mice. This complex is usually used with alum as an adjuvant, which can enhance humoral response but inhibits cell-mediated immune responses. To improve the immunogenicity of HBsAg-anti-HBs, we immunized mice with a combination of this immunogenic complex and pCMVHBs, a plasmid encoding HBsAg, or the vector pCMV. Both plasmids enhanced the anti-HBs response induced by the immunogenic complex. We found 20 microg of plasmid or vector enhanced the anti-HBs response in all mice, whereas 1 microg was less effective. Splenocytes from different immunized groups were stimulated with HBsAg in vitro, and the highest level of IL-2 detected in the supernatant was found in mice immunized with HBsAg-anti-HBs plus pCMVHBs. A plasmid (pcDNA3c191) encoding core protein of hepatitis C virus (HCV) was used as an adjuvant to the immunogenic complex. A preliminary result showed that pcDNA3c191 not only enhanced the immunogenicity of HBsAg-anti-HBs, but also induced anti-HCV core antibodies. Immunization using a plasmid DNA encoding one viral antigen in combination with antigen and antibody complex of another microbial origin could be a new approach to the development of multivalent vaccines.