In the spleen, exogenous antigen is preferentially presented by CD8α+CD11b– DC to CD8 T cells and by CD8α–CD11b+ DC to CD4 T cells. However, it is not yet clear whether the same rule applies to other secondary lymphoid organs. To address this issue, we first classified secondary lymphoid tissues into three categories based on the expression pattern of CD8α and CD11b in C57BL/6 and BALB/c mice: (a) spleen, (b) mesenteric lymph node (MLN) and (c) other peripheral lymph nodes (PLN). We then analyzed the OVA-specific T cell-stimulating capacity of each DC subset after intravenous injection with soluble OVA. Our results show that, regardless of tissue origin, CD8α–CD11b+ DC generally present OVA to CD4 T cells, a finding that held true as well for CD8α+CD11b+ DC in PLN. In striking contrast, CD8α+CD11b– DC in spleen, CD8α–CD11b+ DC in MLN and CD8α+CD11b+ DC in PLN mainly cross-present OVA to CD8 T cells in their respective tissues. Of note, CD8α–CD11b+ DC in MLN and CD8α+CD11b+ DC in PLN present OVA to both CD4 T and CD8 T cells. Therefore, the antigen-presenting capacity of each distinct DC subset is determined by its anatomic environment in combination with its surface phenotype.