Human papillomaviruses (HPVs) are small DNA tumor viruses, a subset of which is closely associated with the development of cervical cancer. The viral E6 and E7 open reading frames encode multifunctional proteins that bind respectively to the p53 protein and to the product of the retinoblastoma tumor-suppressor gene. In this study we demonstrate that the HPV-16 E6 gene cooperates with EJ-ras to immortalize primary cultures of mouse kidney epithelial cells. HPV-16-immortalized cell lines expressing E6 but not E7 contained low levels of wild-type p53 protein. In contrast, those cells immortalized by EJ-ras alone contained elevated p53 protein levels, and were shown to contain a mutation in the gene. These results suggest that activating mutations in the p53 gene can functionally substitute for HPV-16 E6 in transforming primary cells.