Geldanamycin (GA), a heat-shock protein (HSP) 90 inhibitor, induces degradation of HSP90 client proteins, which may promote the presentation of degradation peptides with major histocompatibility complex class I on cancer cells. We hypothesized that GA may enhance the efficacy of DNA vaccination, and investigated the therapeutic effect of the combination of GA and a DNA vaccine against HSP90 clients p185(neu) and Met. The efficacy of various doses of GA combined with an N-terminal neu (N'-neu) DNA vaccine was investigated in a transplanted tumor constitutively overexpressing endogenous p185(neu). Low-dose (2.5 mug) but not high-dose (10 microg) GA enhanced the effect of N'-neu DNA vaccination on the inhibition of murine bladder tumor-2 tumors in syngeneic C3H mice. Anti-p185(neu) antibody titers were similar among all treated groups. Significantly increased infiltrations of CD8(+) T cells and NK cells were observed at tumor sites. GA sensitized tumor cells to the cytotoxic effects of lymphocytes. Depletion of CD8(+) T cells eliminated most of the therapeutic efficacy; in contrast, depletion of CD4(+) T cells enhanced the therapeutic efficacy. A similar enhancing effect was observed for the combination of GA and a DNA vaccine targeting the Met oncogene. Our results support the use of combination of GA and DNA vaccination against GA-targeted proteins.