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The aim of this study was to localize and evaluate the role of Toll-like receptor 2 (TLR2) in the endometrium and cervix of bitches at different stages of the oestrous cycle and in bitches with pyometra. Sixty-seven nulliparous dogs, ranging in age from 1 to 13 years, were allocated amongst five groups (pro-oestrus; n = 7, oestrus; n = 10, dioestrus; n = 16, anoestrus; n = 11, pyometra; n = 23). Blood samples were collected for the measurement of progesterone concentration. The mean progesterone concentration was analysed as a parameter for validating the stage of the oestrous cycle in bitches. Tissues collected from uterine horn and cervix were fixed in 4% paraformaldehyde for immunohistochemical examination of TLR2. The expression of TLR2 was assessed semi-quantitatively. No pathological changes were found in the uterine samples of healthy dogs. In bitches with pyometra, the glandular epithelium expressed TLR2 more intensely than the surface epithelium. The expression of TLR2 in the glandular epithelium was also significantly higher in healthy dogs at oestrus, dioestrus and dogs with pyometra compared with anoestrous dogs (p < 0.01). The expression of TLR2 in the stroma was not observed in the group of healthy dogs at all stages. The surface epithelium of cervix in dogs with pyometra expressed TLR2 significantly more intensely than did the stoma, whereas the expression of TLR2 during oestrus and dioestrus was absent in the stroma of cervix. This study provides the first report of immunohistochemical localization of TLR2 in the canine reproductive tract. In the present study, TLR2 was expressed in endometrial epithelium but was absent in the endometrial stroma of healthy dogs at all oestrous cycle stages. These findings suggest differential expression of TLR in endometrial cells. On the other hand, the lack of TLR2 in the stroma of healthy uteri of dogs may predispose to infection from the invading pathogens once the epithelial cells have been destroyed by the pathogens, especially Gram-positive bacteria.