The proliferating cell nuclear antigen (PCNA) is essential for DNA replication of mammalian cells and their small DNA tumor viruses. The mechanism of the cell cycle-dependent regulation of the human PCNA promoter is not clear despite extensive investigations. In this report, we employed organotypic cultures of primary human keratinocytes, which closely resemble native skin comprising both proliferating and postmitotic, differentiated cells, to examine the cell cycle-dependent regulation of the human PCNA gene (hPCNA) in the absence or presence of the human papillomavirus type 18 (HPV-18) E7 protein. HPV-18 E7 promotes S phase re-entry in post-mitotic differentiated keratinocytes by abrogating the transcription repression of E2F transcription factors by the retinoblastoma susceptibility protein, pRb. We demonstrated that E7 reactivated the transcription of the endogenous hPCNA in differentiated keratinocytes. In contrast, with or without E7, the expression of a transduced hPCNA promoter-driven reporter did not correlate with that of the endogenous hPCNA gene in either proliferating or differentiated cells. Moreover, in Chinese hamster ovary and L-cells, HPV E7 and the adenovirus E1A protein repressed the transduced hPCNA promoter, but both activated an extended promoter construct spanning the first intron. Mutations of two E2F sites in the intron reduced the basal activity and abolished the response to E7 or E1A. Promoter repression or activation required the CR2 domain of E7 and, to a lesser extent, CR1 as well. However, in organotypic cultures, this extended promoter construct failed to recapitulate the cell cycle-dependent regulation of the endogenous hPCNA gene. Only when a full-length Myc-tagged hPCNA spanning the 5′ promoter and all exons and introns was used was the native pattern of expression largely restored, indicative of the complexity of its regulation.