Epithelial cells immortalized by human papillomaviruses have premalignant characteristics in organotypic culture. Three HPV-16--and four HPV-18--immortalized human foreskin keratinocyte cell lines were analyzed on organotypic epidermal raft cultures at various passage levels. This culture system allowed normal cultured keratinocytes to stratify and differentiate in a manner similar to normal epidermis. All seven HPV-immortalized cell lines displayed epidermal morphologies on organotypic cultures, which were clearly abnormal and resembled premalignant lesions in vivo. Features of premalignant lesions that were shared by all of the HPV-immortalized cell lines included disorganized tissue architecture, mitotic cells present throughout the living layers of the epidermal sheet, abnormal mitoses, enlarged nuclei, and variable cell size and shape. Most HPV-immortalized cell lines were stable in terms of epidermal morphology with long-term passage in culture. Two of the HPV-18--immortalized cell lines, however, lost all morphologically apparent terminal squamous differentiation potential after long-term passage in monolayer culture. These results strongly support the idea that immortalization of squamous epithelial cells in culture by HPV-transforming genes generates a morphologically premalignant cell. 