The aim of this study was to determine the types of Toll-like receptors (TLRs) expressed in the hen oviduct, and to confirm that vaginal tissue expressing TLR4 responds to lipopolysaccharide (LPS). Healthy laying hens were intravenously or intravaginally injected with LPS, PBS or untreated. The expression pattern of TLRs in the whole oviduct and the effects of LPS on TLR4 and IL-1beta in the vagina were examined by semi-quantitative RT-PCR. The population of cells containing TLR4 protein was examined by immunohistochemistry. The expression of 6 types of TLRs (TLR1 type 2, TLR2, TLR3, TLR4, TLR5 and TLR7) were identified in all segments of the oviduct. The densities of PCR products for each TLR showed a tendency to be greater in the vagina than in the magnum and isthmus. Immunoreactive TLR4 was localized in the epithelial cells and leukocytes in the isthmus, uterus and vagina. Intravenous injection with LPS increased the TLR4 expression and the population of TLR4-immunopositive cells in the vagina. Intravaginal injection with LPS resulted in the increase of leukocytes in the mucosal tissues in association with an increase of TLR4 expression and TLR4-immunopositive cells in the vagina. The IL-1beta expression was also enhanced in a similar manner to that of TLR4. These results suggest that the hen oviduct expresses at least 6 types of TLRs including TLR4 with a greater expression in the vagina. Vaginal tissue expressing TLR4 responds to LPS and in turn upregulate cellular functions to synthesize cytokines. Such expression and functions of TLRs may play an essential role in oviductal innate immunity for host defense.