The human papillomavirus (HPV) type 18 E6 gene cooperates with activated Ha-ras to immortalize primary mouse cells in culture. Using a plasmid where HPV18 E6 expression is regulated by the glucocorticoid inducible MMTV LTR, we have generated immortalized cell lines in which the continued expression of E6 was necessary for maintenance of the transformed phenotype. In the absence of exogenously added hormone these cells were found to arrest in G0/G1. Furthermore, we demonstrate that the effects of E6 were essentially p53 independent and therefore define a novel function by which E6 is able to modulate cell proliferation. In addition, when the E6 dependent cells were maintained under conditions of prolonged growth arrest by the removal of E6, revertant cells were isolated which were no longer dependent on E6 expression for continued proliferation. These revertant cells were found to have acquired a mutation in the cellular gene p53, suggesting that certain p53 mutations are dominant over an E6 requirement in this assay.