Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns (PAMPs) and enable innate immune responses. Although TLR9 has been previously considered to be expressed only in immune cells, there is now increasing evidence that TLR9 expression is present in nonimmune cells as well. In this study, we undertook to determine whether TLR9 expression was associated with disease progression in cervical neoplasia. TLR9 expression was evaluated by immunohistochemistry in 55 formalin-fixed paraffin-embedded cervical tissues; nine normal cervical specimens, 10 low-grade cervical intraepithelial neoplasias (CINs), 12 high-grade CINs, and 24 invasive squamous cell carcinomas (ISCCs). In addition, TLR9 expression was evaluated, at the RNA level, in fresh frozen cervical carcinoma tissues by real-time quantitative RT-PCR. Immunohistochemical staining showed that TLR9 expression was undetectable (55.6%) or weak (44.4%) in normal cervical squamous epithelial tissues, however, variable staining was observed in the basal layer of all normal endocervical glands. TLR9 expression, which was mainly observed as cytoplasmic staining, gradually increased in accordance with the histopathological grade in the following order: low-grade CIN < high-grade CIN < ISCC (P < 0.001), and in particular, was moderate to strong in 70% of ISCC cases. Furthermore, real-time quantitative RT-PCR revealed that TLR9 expression, in tumors, was significantly enhanced compared to normal cervical tissues (P = 0.012). These results suggest that TLR9 may play a significant role in tumor progression of cervical neoplasia and may represent a useful marker for malignant transformation of cervical squamous cells. © 2007 Wiley-Liss, Inc.