High-risk human papillomaviruses (HPV) encode two oncogenes, E6 and E7, expressed in nearly all cervical cancers. In vivo, HPV-16 E7 has been shown to induce multiple phenotypes in the context of transgenic mice, including cervical cancer. E7 is a multifunctional protein known best for its ability to inactivate the tumor suppressor pRb. To determine the importance of pRb inactivation by E7 in cervical cancer, we pursued studies with genetically engineered mice. E7 expression in estrogen-treated murine cervix induced dysplasia and invasive cancers as reported previously, but targeted Rb inactivation in cervical epithelium was not sufficient to induce any cervical dysplasia or neoplasia. Furthermore, E7 induced cervical cancer formation even when the E7-pRb interaction was disrupted by the use of a knock-in mouse carrying an E7-resistant mutant Rb allele. pRb inactivation was necessary but not sufficient for E7 to overcome differentiation-induced or DNA damage-induced cell cycle arrest, and expression patterns of the E2F-responsive genes Mcm7 and cyclin E indicate that other E2F regulators besides pRb are important targets of E7. Together, these data indicate that non-pRb targets of E7 play critical roles in cervical carcinogenesis.