This study was undertaken to explore the expression of keratins in the hamster cheek pouch carcinogenesis model, using monospecific keratin antibodies and a technique that allows immunoblotting analysis of tissues embedded in paraffin. Changes in keratin expression were correlated with histopathological changes and with the expression of the enzyme gamma-glutamyl transpeptidase. The right cheek pouch of 20 male golden Syrian hamsters was treated with 0.5% 7,12-dimethylbenz[a]anthracene for 16 weeks. As previously described by other laboratories, this treatment resulted in hyperplastic and dysplastic lesions and benign and malignant tumors. The keratins assayed in this study were K14 (Mr 55,000), K1 (Mr 67,000), and K13 (Mr 47,000). The normal hamster cheek pouch epithelium expressed K14 in the basal layer and K13 in the suprabasal and differentiated layers, whereas K1 was not detected by either immunohistochemistry or immunoblotting. Concomitant with 7,12-dimethylbenz[a]anthracene-induced hyperplasia, there were some topographical alterations in the distribution of K14. In this case, K14 was no longer restricted to the basal layer but was also expressed in differentiated cells. The same pattern was also observed in dysplastic lesions and in squamous cell carcinoma. Furthermore, expression of the K13 differentiation-associated keratin was preserved in this hyperplastic epithelium during all the stages of carcinogenesis, including either anaplastic or differentiated areas. In contrast, after 2 weeks of 7,12-dimethylbenz[a]anthracene treatment, K1 expression started as a weak and patchy pattern in suprabasal cells, becoming stronger and more homogeneous at 8 and 16 weeks of treatment. However, K1 was almost absent in squamous cell carcinoma, where only small very well differentiated areas were stained. We also observed gamma-glutamyl transpeptidase-positive foci in earlier stages of carcinogenesis, concomitant with the expression of the K1 keratin. However, it was not possible to find a perfect topographical correspondence between the two events. Alterations in the pattern of keratin expression appear to be a common feature during the development of squamous cell carcinoma in different systems and could be an excellent tool to study carcinogenesis and chemoprevention.