Plasmid DNA encoding the flagella protein (flagellin) was used as a vaccination candidate for the evaluation of its immunogenicity and for protection against infection with Burkholderia pseudomallei. Firstly, flagellin encoding plasmid DNA was injected into Balb/c mice intramuscularly and this elicited both a humoral and a cellular immune response. Total IgG production and the clonal expansion of the spleen cells increased in response to flagellin. The IgG subclass response exhibited a dominance of IgG2a over IgG1 in the sera. In addition, IFN-gamma-secreting cells in the spleen were substantially increased. Furthermore, the anti-B. pseudomallei activity of the peritoneal exudate cells was evaluated by a Transwell tissue-culture plate system where the macrophage-activating related cytokines in upper chamber were allowed to cross the plate's membrane and stimulate the activation of peritoneal exudate cells in lower chamber. Our results indicated that the activated peritoneal exudate cells were able to restrict the growth of B. pseudomallei in vitro. Indeed, subsequent intravenous challenge of the vaccinated Balb/c mice with 10(5)CFU of B. pseudomallei resulted in the number of bacterial cells detected in liver and/or spleen being significantly reduced in the flagellin plasmid DNA vaccinated mice. At 7 days subsequent to infection of B. pseudomallei, 5/6 (83%) of flagellin plasmid DNA vaccinated mice had survived. We suggest that plasmid DNA-encoding flagellin might be useful as a potential immunization route for the future development of a vaccine against melioidosis in related animals.