Intramuscular (i.m.) injection of mice with plasmid DNA expression vectors containing all or part of the hepatitis B virus (HBV) gene encoding the envelope proteins induces a strong humoral response to the HBV surface antigen (HBsAg) which is sustained for up to 74 weeks without boost. After a single i.m. injection of 100 micrograms DNA, antibodies to HBsAg (anti-HBs) reach ELISA titers of 4 x 10(4) in C57BL/6 mice and 10(4) in BALB/c mice, or somewhat less in older mice. Although antibody levels induced by a single injection of DNA do not diminish significantly over time, they can be further increased 10-200-fold by boosting with a second injection of DNA or an injection of recombinant HBsAg protein. Prior injection of DNA does not affect the strength or timing of the boosting effect, suggesting that there is no immune response against the vector itself. Boosting with a second injection of DNA is possible even in BALB/c mice, which are known to have a strong cytotoxic T-lymphocyte response against an epitope on the major HBV envelope protein, indicating that possible destruction of newly transfected muscle fibers is not so quick and efficient as to abort the boosting effect. A single injection of DNA results in a stronger and longer lasting humoral response than does a single injection of recombinant protein.