Molecular events occurring with high-risk human papillomavirus (HPV)-associated dysplastic differentiation of cervical epithelial cells are largely unknown. This study used differential display PCR to identify expression changes between nondifferentiating monolayer and differentiated organotypic (raft) cultures of W12 keratinocytes. These cells were originally derived from a clinical biopsy of HPV 16-positive dysplastic cervical epithelium and retain high-risk HPV 16 and the ability to differentiate, albeit with dysplastic morphology. Using this model system we identified 84 genes with changed expression during dysplastic differentiation. Most (70/84, approximately 80%) were down-regulated with differentiation, consistent with a restriction of expression during terminal differentiation. Twenty-two genes had no known function and 6 novel expressed sequence tags were identified among this group. Of the 62 genes with known functions, 25 belonged to transcription-, translation-, and posttranslation-related categories and 30 had functions associated with neoplastic initiation/progression, calcium signaling, epithelial differentiation, and structure remodeling. Some of the genes with altered expression identified in this model of dysplastic differentiation may be useful biomarkers for early detection of cervical neoplasia and other HPV-associated oropharyngeal and anogenital cancers.