Intramuscular delivery of plasmid DNA encoding vaccine antigens induces systemic immune responses in small animals but is less effective in inducing strong responses in larger animals species. We investigated if anatomic distribution of DNA could enhance responses to decreasing DNA doses. We show that anatomic distribution of low-dose DNA improves cell-mediated and humoral immune responses in mice. Anatomic distribution of low-dose DNA provided a dose-sparing effect for both cell-mediated immunity and humoral responses. Enhanced systemic responses correlate directly with recruitment of antigen-specific CD8+ T-cells in the regional lymph nodes. These results have implications for DNA vaccination in humans.