Commensal gut bacteria have potent effects on the immune system, which are partially mediated by intestinal dendritic cells (DC). Distinct commensals confer different properties to in vitro-generated DC. The aim of the present study was to reveal strain-dependent maturation patterns in primary DC. To this end, we compared the response of mouse Peyer's patch (PP) DC, mesenteric lymph node (MLN) DC and spleen DC to the commensal bacteria, Bifidobacterium longum Q46, Lactobacillus acidophilus X37 and Escherichia coli Nissle 1917. Bacterial maturation of DC occurred independently of tissue origin. Expression of CCR7 and CD103 on the surface of MLN DC, necessary for the induction of gut-homing regulatory T cells, increased with stimulation by Gram-positive commensals. Bacteria-dependent cytokine production (IL-6, IL-10 and TNF-alpha) was similar in spleen and MLN DC, and contaminant cells in these DC preparations produced IFN-gamma in response to L. acidophilus. In contrast, PP DC produced IL-6 only in response to E. coli, little IL-10 and no TNF-alpha, and this low cytokine production was not due to inhibition by IL-10 or TGF-beta. Bifidobacteria downregulate IL-6, TNF-alpha and IL-12 production induced in in vitro-generated DC by L. acidophilus. Similar inhibition was observed in splenic DC, but not in MLN DC. MLN cells responded to bacterial stimulation with higher IFN-gamma production than spleen cells, possibly due to the presence of more responsive natural killer cells. Commensal bacteria therefore play specific roles in the gut immune system distinguishable from the effect they would have if recognized by the systemic immune system.