The human papillomavirus type 18 E7 protein subverts the pRb/E2F pathway to promote S-phase reentry by postmitotic, differentiated primary human keratinocytes in support of viral DNA amplification. We prepared a panel of HPV-18 E7 mutations in pRb binding or in casein kinase ll (CKll) phosphorylation. Our results showed that the ability of E7 binding to pRb correlated with the activation of DNA polymerase α or cyclin E to various extents in differentiated keratinocytes of organotypic cultures but was insufficient to induce the proliferating cell nuclear antigen. Proteins mutated in the CKll recognition sequence or in one or both serine substrates (S32 and S34) bound pRb in vitro, but only those with negative charges at these two residues induced proliferating cell nuclear antigen effectively. Nevertheless, unscheduled cellular DNA synthesis occurred very inefficiently relative to the wild-type E7, if at all. Thus, both pRb binding and CKll phosphorylation of E7 are critical for activating cellular genes essential for S-phase entry.