The patterns of expression of keratins K1 and K8, filaggrin, and the proliferation-associated protein, proliferating cell nuclear antigen (PCNA), were studied in normal and human papillomavirus (HPV) 16 or 18 immortalized keratinocyte cell lines grown on organotypic raft cultures. Normal keratinocytes produced an epithelial sheet that closely resembled epidermis in vivo, characterized by lack of K8 expression, PCNA expression restricted to the basal layer, and K1 and filaggrin expression in the suprabasal layers. Although morphologically abnormal in many respects, some HPV-immortalized cell lines produced cornified epithelial layers and approximated the normals in their patterns of expression of keratins and filaggrin. Other HPV-immortalized cell lines produced poorly differentiated epithelial layers that were characterized by loss of filaggrin expression, and the single tumorigenic cell line, 18-11, was distinguished by abundant K8 expression. All of the HPV-immortalized cell lines were distinguished from normal keratinocytes by a common pattern of full-thickness PCNA expression in the epithelial layers they produced, suggesting that maintenance of the proliferative state may be an important contribution made by HPV 16 or 18 sequences toward the production of a malignant phenotype.