Proteomic analysis of squamous cell carcinoma of the uterine cervix was performed using total protein from archival formalin-fixed, paraffin-embedded tissues. A wide range of proteins with molecular weights of 10 to greater than 200 kd was extracted from formalin-fixed, paraffin-embedded tissues using a recently developed protocol based on the heat-induced antigen retrieval technique. The extracted proteins from normal squamous epithelium (n = 53) and squamous cell carcinoma (n = 21) were fluorescently labeled and separated using 2-dimensional gel electrophoresis. We identified 728 differentially expressed proteins, with 144 up-regulated and 584 down-regulated as compared with normal squamous epithelial tissue samples. Nine proteins showing pronounced up-regulation in squamous cell carcinoma were analyzed on liquid chromatography-tandem mass spectrometry. Among the candidate proteins identified, minichromosome maintenance 8, a disintegrin and metalloproteinase domain 18, and heat shock protein 27 were analyzed in Western blotting, resulting in significant overexpression of heat shock protein 27 in squamous cell carcinoma over normal mucosa (P < .05). Furthermore, immunostaining revealed heat shock protein 27 overexpression not only in squamous cell carcinoma but in various stages of cervical intraepithelial neoplasia (grades 1-3, n = 90), including dysplasia and carcinoma in situ. The expression levels of heat shock protein 27 in cervical intraepithelial neoplasia grades 1 to 3 and squamous cell carcinoma were significantly higher than that in normal mucosa (P < .05). In the neoplastic lesions, heat shock protein 27 expression levels in cervical intraepithelial neoplasia grade 3 and squamous cell carcinoma were significantly higher than that in cervical intraepithelial neoplasia grade 1 (P < .05). These results may suggest a role of heat shock protein 27 in tumor development and progression in the cervical intraepithelial neoplasia-squamous cell carcinoma sequence. Future experiments using formalin-fixed, paraffin-embedded tissue-based proteomic analysis will be a powerful tool for various pathologic studies.