An adjuvant role for certain short bacterial immunostimulatory DNA sequences (ISSs) has recently been proposed on the basis of their ability to stimulate T helper-1 (Thl) responses in gene-vaccinated animals. We report here that noncoding, ISS-enriched plasmid DMAs or ISS oligonucleotides (ISS-ODNs) potently stimulate immune responses to coadministered antigens. The ISS-DNAs suppress IgE synthesis, but promote IgC and interferon-γ (IFN-γ) production. They furthermore initiate the production of IFN-γ, IFN-α, IFN-β, and interleukins 12 and 18, all of which foster Thl responses and enhance cell-mediated immunity. Consideration should be given to adding noncoding DNA adjuvants to inactivated or subunit viral vaccines that, by themselves, provide only partial protection from infection.