Two activities of human papillomavirus type 16 E6 (HPV16 E6) are proposed to contribute to the efficient immortalization of human epithelial cells: the degradation of p53 protein and the induction of telomerase. However, the requirement for p53 inactivation has been debated. Another E6 target is the hAda3 protein, a p53 coactivator and a component of histone acetyltransferase complexes. We have previously described the role of hAda3 and p53 acetylation in p14ARF-induced human mammary epithelial cell (MEC) senescence (P. Sekaric, V. A. Shamanin, J. Luo, and E. J. Androphy, Oncogene 26:6261-6268, 2007). In this study, we analyzed a set of HPV16 E6 mutants for the ability to induce hAda3 degradation. E6 mutants that degrade hAda3 but not p53 could abrogate p14ARF-induced growth arrest despite the presence of normal levels of p53 and efficiently immortalized MECs. However, two E6 mutants that previously were reported to immortalize MECs with low efficiency were found to be defective for both p53 and hAda3 degradation. We found that these immortal MECs select for reduced p53 protein levels through a proteasome-dependent mechanism. The findings strongly imply that the inactivation of the p14ARF-p53 pathway, either by the E6-mediated degradation of p53 or hAda3 or by cellular adaptation, is required for MEC immortalization.