Tyrosinase-related protein-2 (TRP2) is a weak antigen expressed in murine and human melanomas. Induction of antibody (Ab) response and T-cell immunity toward TRP2 with DNA plasmid vaccines has not been efficient to date. Recent studies have suggested that a chemokine ligand for the CCR7 (CCL21) present on T-cells and dendritic cells is important in activating and regulating immunity. We investigated the effectiveness of CCL21 as an adjuvant with an HVJ anionic liposomal TRP2 DNA (plasmid) vaccine to enhance anti-TRP2 Ab, cytokines, delayed-type hypersensitivity, T-cell responses, and tumor protection against B16 melanoma cells. Induction of anti-TRP2 immunity depended mainly on cell-mediated immunity, which was regulated by timing and route of CCL21 administration with DNA vaccine. The optimum protocol was to administer CCL21 im 24 h before DNA vaccine at the same vaccination site. Two vaccinations (prime/boost) were essential for induction of strong anti-TRP2 cell-mediated immunity. CCL21 administration 3 days before or 24 h after DNA vaccine, simultaneous with DNA vaccine, or at different sites (iv, opposite leg) was not effective. This study demonstrated that CCL21 was an effective adjuvant to enhance TRP2-specific immunity induced by a plasmid DNA cancer vaccine.