Recently we have shown that a bacterial flagellin, Vibrio vulinifiucs FlaB(Vv-FlaB), has strong adjuvant activity to induce protective immune response. In order to investigate the adjuvanticity of Vv-FlaB, we prepared highly purified recombinant protein by using an intein fusion protein purification system. However, in the process of the purification, we unexpectedly encountered a contamination with a 70 kDa protein. We proved the 70 kDa protein as the heat shock protein 70 (HSP70) by Western blotting. Unfortunately, it was reported that the HSP70 has a strong adjuvanticity. In this study we investigated the role of contaminating HSP70 on the Vv-FlaB-mediated adjvanticity. We separated Vv-FlaB and HSP70 by using a high performance protein purification chromatography and compared adjuvant activities of Vv-FlaB, HSP70 and Vv-FlaB/HSP70 mixture. Using an intranasal immunization mouse model, we observed hat co-administration of the flagllin with tetanus toxoid (TT) induced significantly enhanced TT-specific antibody (Ig) responses. However contaminating doses of HSP70 id not affect the adjuvanticity of Vv-FlaB and furthernmore HSP70 alone did not enhance TT-specific Ig response and protective immunity against lethal challenge with tetanus toxin. These results show that the HSP70 contaminating Vv-FlaB preparations did not affect the adjuvanticity of Vv-FlaB.