Dendritic cells (DCs) are professional antigen-presenting cells, well equipped to initiate an immune response. Currently, tumor antigen-derived peptide loaded DCs are used in clinical vaccination in cancer patients. However, the optimal dose and route of administration of a DC vaccine still remain to be determined. Using indium-111-labeled DCs, we investigated whether the route of administration does affect the biodistribution of DCs in lymphoid organs and whether it influences the outcome of DC vaccination in the B16 mouse melanoma tumor model. The results demonstrate that i.v. injected DCs mainly accumulate in the spleen, whereas s.c. injected DCs preferentially home to the T-cell areas of the draining lymph nodes. Using tyrosinase-related protein-2-derived peptide-loaded DC vaccination in a fully autologous B16 melanoma tumor model, we observed a delay in tumor growth, improved survival as well as increased antitumor cytotoxic T-cell reactivity after s.c. vaccination as compared to i.v. vaccination. These data demonstrate that optimal induction of antitumor reactivity against the autologous melanocyte differentiation antigen tyrosinase-related protein-2-derived peptide occurs after s.c. vaccination and correlates with the preferential accumulation of DCs in the T-cell areas of lymph nodes.