Toll-like receptors are key elements in pathogen recognition by the host immune system. Although the expression pattern and functions of Toll-like receptors have been studied in a variety of cytokine-induced dendritic cells, it remains unknown whether Toll-like receptor stimulation influences maturation and cytokine production of authentic Langerhans cells. We purified murine epidermal Langerhans cells along with splenic dendritic cell using a panning method. Langerhans cells expressed Toll-like receptor 2, 4, and 9 but not 7, the pattern of which suggests Langerhans cells are the closest to one of the murine dendritic cell lineage, CD11c+11b+8 alpha-4-. Then we stimulated Toll-like receptor 2, 4, and 9 with the corresponding ligand, Staphylococcus aureus Cowan 1, lipopolysaccharide, and CpG, and found that all of these stimuli upregulated expression of B7-1 and B7-2 in splenic dendritic cells but not in Langerhans cells. As in human Langerhans cells, stimulation of murine Langerhans cells with Staphylococcus aureus Cowan 1, lipopolysaccharide, and CpG overall resulted in T helper 1-polarizing cytokine production (namely, induction of IL-12p40 and inhibition of TARC (thymus and activation-regulated chemokine)/CCL17). Exceptionally, lipopolysaccharide exhibited no effect on IL-12p40 production by Langerhans cells and inhibited IL-12p40 production by splenic dendritic cells. These results may represent the functional heterogeneity between Langerhans cells and splenic dendritic cells, and are important for better understanding of innate immunity to bacterial infections differentially regulated in the skin and spleen. MeSH terms: Toll-like receptors, Langerhans cells, dendritic cells.