Human papillomaviral (HPV) origin-containing plasmids replicate efficiently in human 293 cells or cell extracts in the presence of HPV origin-recognition protein E2 and replication initiation protein E1, whereas cervical carcinoma-derived, HPV-18-positive HeLa cells or cell extracts support HPV DNA replication poorly. We recently showed that HPV-11 E1 interacts with cyclin/cyclin-dependent kinase (cdk) complexes through an RXL motif and is a substrate for these kinases. E1 mutations in this motif or in candidate cdk phosphorylation sites are impaired in replication, suggesting a role for cdks in HPV replication. We now demonstrate that one limiting activity in HeLa cells is cyclin E/CDK2. Purified cyclin E/CDK2 or cyclin E/CDK3 complex, but not other cdks, partially complemented HeLa cell extracts. Cyclin E/CDK2 expression vectors also enhanced transient HPV replication in HeLa cells. HeLa cell-derived HPV-18 E1 protein is truncated at the carboxyl terminus but can associate with cyclin E/CDK2. This truncated E1 was replication-incompetent and inhibited cell-free HPV replication. These results indicate that HeLa cells are phenotypically limiting in cyclin E/CDK2 for efficient HPV replication, most likely due to sequestration by the endogenous, defective HPV-18 E1 protein. Further analyses of the regulation of HPV E1 and HPV replication by cyclin E may shed light on the roles of cyclin E/CDK2 in cellular DNA replication.