Enhancement of DNA vaccine immunogenicity is a current topic of high priority in the field of applied immunology, especially as a means of controlling HIV infection. The adjuvant effect of Ubenimex (UBX), an anti-cancer immunomodulator, on a DNA AIDS vaccine which we developed was examined in a murine model. UBX was formulated into a preparation containing DNA plasmids encoding env and rev genes of HIV-1 strain III(B), and was inoculated intramuscularly into BALB/c mice. The sera obtained with this mixture had 2(3)-2(5) times higher specific IgG titres than those obtained without the use of the adjuvant. UBX also elicited both a stronger HIV-1-specific DTH reaction, as measured by the footpad swelling test, and stronger cytotoxic T lymphocyte activity, as assayed by the 51Cr-release method, compared with responses using DNA alone. The cytokine secretion profile of restimulated immune lymphoid cells showed that UBX raised IL-2 and interferon-gamma levels and decreased IL-4 production. HIV-1-specific immunoglobulin subtype analysis demonstrated that UBX stimulated IgG2a production but suppressed synthesis of IgG1 and IgE. These results indicate that activation of the T-helper type 1 subset was induced by UBX, suggesting a mechanism of immunomodulation mediated by this agent. We conclude that UBX acts as an immunologic adjuvant for DNA vaccination against HIV-1. UBX may be a suitable adjuvant for clinical use because of its lack of antigenicity and low toxicity.