The productive program of human papillomaviruses occurs in differentiated squamous keratinocytes. We have previously shown that vegetative amplification of the HPV-18 genomic plasmid initiates in G2-arrested spinous cells in organotypic raft cultures of primary human keratinocytes (PHKs), as marked by their abundant cytoplasmic cyclin B1 (Wang et al., 2009. Genes Dev. 23: 181-194). We now demonstrate that the E7 protein, which induces S phase reentry in suprabasal cells, also induces cellular environments conducive for prolonged G2 phase. Western blots and indirect immunofluorescence assays were used to probe for host proteins known to control G2/M progression. E7 expression induced cytoplasmic cyclin B1 and cdc2 in the suprabasal cells. In addition, there were elevated Wee1 and Myt1 proteins that are responsible for inhibitory phosphorylation of cdc2 on Threonine 14 and Tyrosine 15. Indeed, the elevated cdc2 had inactivating phosphorylation on T14 or Y15, and possibly both. Moreover, in cells harboring cytoplasmic cyclin B1 or cdc2, there was an accumulation of elevated cdc25C phosphorylated on Serine 216, an inactive form unable to dephosphorylate and activate cdc2. Taken together, this study has revealed the mechanisms by which E7 induces prolonged G2 phase in the differentiated cells following S phase induction to enable viral DNA amplification. Similar results were observed in PHK raft cultures in which the fully productive life cycle of HPV-18 took place.