mRNA transcription, DNA amplification, and progeny production of human papillomaviruses (HPVs) are closely linked to squamous epithelial differentiation in patient papillomas. Because suprabasal, differentiated keratinocytes have exited the cell cycle for days or weeks and because vital DNA synthesis requires the host DNA replication machinery, HPVs must have a mechanism to reactivate the essential host genes. In this study, we show via acute recombinant retrovirus infection that an intact E7 gene of either high- risk or of low-risk HPV genotypes, under the control of its respective native enhancer-promoter, induced proliferating cell nuclear antigen (PCNAs) expression in the suprabasal cells of epithelial raft cultures of primary human foreskin keratinocytes (PHK). The cellular differentiation program was unaltered by the vital oncoprotein; it was essential for high HPV promoter activity. Furthermore, extensive host chromosomal DNA replication took place in differentiated cells of HPV-18 E7-expressing raft cultures and of patient laryngeal papillomas caused by HPV-6. These results indicate that the main function of the E7 protein is to reactivate host DNA replication machinery to support vital replication in differentiated, noncycling cells.