A plasmid vector encoding the cholera toxin B subunit (pCtB) was evaluated as an intradermal genetic adjuvant for a model DNA vaccine expressing the human papillomavirus type 16 L1 capsid gene (p16L1) in mice. p16L1 was coadministered with plasmid pCtB or commercial polypeptide CtB as a positive control. Coadministration of pCtB induced a significant increment of specific anti-L1 immunoglobulin A (IgA) antibodies in cervical secretions (P < 0.05) and fecal extracts (P < 0.005). Additionally, coadministration of pCtB enhanced the production of interleukin-2 and gamma interferon by spleen cells but did not affect the production of interleukin-4, suggesting a Th1-type helper response. Furthermore, improved CD8+ T-cell-mediated cytotoxic activity was observed in mice vaccinated with the DNA vaccine with pCtB as an adjuvant. This adjuvant effect was comparable to that induced by the CtB polypeptide. These results indicate that intradermal coadministration of pCtB is an adequate means to enhance the mucosa-, Th1-, and CD8+-mediated cytotoxic responses induced by a DNA vaccine.