We have recently demonstrated the presence of three populations of dendritic cells (DC) in the murine Peyer’s patch. CD11b+/CD8α−(myeloid) DCs are localized in the subepithelial dome, CD11b−/CD8α+(lymphoid) DCs in the interfollicular regions, and CD11b−/CD8α−(double-negative; DN) DCs at both sites. We now describe the presence of a novel population of intraepithelial DN DCs within the follicle-associated epithelium and demonstrate a predominance of DN DCs only in mucosal lymphoid tissues. Furthermore, we demonstrate that all DC subpopulations maintain their surface phenotype upon maturation in vitro, and secrete a distinct pattern of cytokines upon exposure to T cell and microbial stimuli. Only myeloid DCs from the PP produce high levels of IL-10 upon stimulation with soluble CD40 ligand− trimer, or Staphylococcus aureus and IFN-γ. In contrast, lymphoid and DN, but not myeloid DCs, produce IL-12p70 following microbial stimulation, whereas no DC subset produces IL-12p70 in response to CD40 ligand trimer. Finally, we show that myeloid DCs from the PP are particularly capable of priming naive T cells to secrete high levels of IL-4 and IL-10, when compared with those from nonmucosal sites, while lymphoid and DN DCs from all tissues prime for IFN-γ production. These findings thus suggest that DC subsets within mucosal tissues have unique immune inductive capacities.