Orally administered antigens often generate immune responses that are distinct from those injected systemically. The role of antigen-presenting cells in determining the type of T helper cell response induced at mucosal versus systemic sites is unclear. Here we examine the phenotypic and functional differences between dendritic cells (DCs) freshly isolated from Peyer's patches (PP) and spleen (SP). Surface phenotypic analysis of CD11c⁺ DC populations revealed that PP DCs expressed higher levels of major histocompatibility complex class II molecules, but similar levels of costimulatory molecules and adhesion molecules compared with SP DCs. Freshly isolated, flow cytometrically sorted 98–100% pure CD11c⁺ DC populations from PP and SP were compared for their ability to stimulate naive T cells. First, PP DCs were found to be much more potent in stimulating allogeneic T cell proliferation compared with SP DCs. Second, by using naive T cells from ovalbumin peptide–specific T cell receptor transgenic mice, these ex vivo DCs derived from PP, but not from SP, were found to prime for the production of interleukin (IL)-4 and IL-10 (Th2 cytokines). In addition, PP DCs were found to prime T cells for the production of much lower levels of interferon (IFN)-γ (Th1) compared with SP DCs. The presence of neutralizing antibody against IL-10 in the priming culture dramatically enhanced IFN-γ production by T cells stimulated with PP DCs. Furthermore, stimulation of freshly isolated PP DCs via the CD40 molecule resulted in secretion of high levels of IL-10, whereas the same stimulus induced no IL-10 secretion from SP DCs. These results suggest that DCs residing in different tissues are capable of inducing distinct immune responses and that this may be related to the distinct cytokines produced by the DCs from these tissues.