Human papillomaviruses (HPVs) are the causative agents of cervical and other cancers. The oncoprotein E7 activates the cell cycle and makes possible replication of the viral genome in differentiating epithelia. The HPV16 late promoter is activated upon cellular differentiation and regulates late gene expression. We investigated the effect of E7 on the late promoter and found that E7 was able to activate the promoter. In contrast, the other known viral transcriptional regulator, E2, had no effect on the late promoter. Promoter activation by E7 occurred despite inhibition of promoter activity by factors involved in the cell cycle, such as cyclin dependent kinases and E2F transcription factors, and by the ability of E7 to disrupt several aspects of cellular differentiation. These results suggest a new role for E7 in the context of the viral life cycle and shed light on the complex regulation of viral gene expression in infected, differentiating epithelia.