More than 25% of head and neck squamous cell carcinomas (HNSCC) and 99% of cervical cancers (CxCa) are positive for high-risk human papillomaviruses (HPVs). Furthermore, the type I tyrosine kinase receptor ErbB-2 is overexpressed in at least 30% of HNSCC and CxCa. Recently, we demonstrated that E6/E7 of HPV type 16 cooperate with ErbB-2 to induce cell transformation of human normal oral epithelial (NOE) cells. This is accompanied by overexpression of cyclin D1 in NOE cells. To determine the role of cyclin D1 in E6/E7/ErbB-2 cooperation, we examined the independent effects of E6/E7 and ErbB-2, and the combined effect of E6/E7 and ErbB-2 in mouse normal embryonic fibroblast (NEF), wild type (wt), and knockout cyclin D1 (D1 À/À ) cells. We report that NEF-wt cells transduced with E6/E7 alone and E6/E7/ErbB-2 together form small and large tumors in nude mice, respectively, as well as different sized colonies in soft agar; whereas ErbB-2 alone elicits neither tumor formation in vivo nor colony formation in soft agar. More importantly, E6/E7, ErbB-2 and E6/E7/ErbB-2 together all fail to induce neoplastic transformation of cyclin D1 À/À cells in vivo and in vitro. Furthermore, using antisense cyclin D1 we completely inhibited tumor and colony formation of NEF-wt-E6/E7 and wt-E6/E7-ErbB-2 as well as human NOE-E6/E7-ErbB-2-transformed cells. These analyses reveal that cyclin D1 is the downstream target of the neoplastic transformation induced by E6/E7 or E6/E7/ErbB-2 cooperation in normal cells. Our data suggest that anti-cyclin D1 therapy may be highly specific in the treatment of all human cancers expressing high-risk HPVs or HPVs/ErbB-2.Oncogene ( Keywords: cyclin D1; E6/E7 of HPV; ErbB-2; cell transformation Head and neck (HN) cancer is the sixth most common neoplasm in the world today in both men and women (Gasco and Crook, 2003). Cervical carcinoma (CxCa) is the second most common cancer among women worldwide and the most frequent female cancer in large areas of the developing world (Clifford et al., 2003). The overall 5-year survival rate of patients diagnosed with HN cancer or CxCa is approximately 60% and has not significantly improved over the past two decades. An important etiological factor is the human papillomavirus (HPV), as roughly 25% of HN cancers and 99% of CxCa are positive for high-risk HPVs (types 16 and 18) (Ke et al., 1999;Gillison et al., 2000;Niv et al., 2000;Moreno et al., 2002). The high-risk HPV E6 and E7 oncoproteins, which are constitutively expressed in these cancers, inactivate the p53 and pRb tumor suppressors, respectively. However, HPV infection alone is not sufficient for neoplastic progression in humans; the HPV-infected cell must undergo additional genetic changes. Moreover, the type I tyrosine kinase receptor ErbB-2 is overexpressed in at least 30% of HN cancers and CxCa (Xia et al., 1997;Wilkman et al., 1998;Ibrahim et al., 1999;Lee et al., 2002). No studies have been performed that address the potential importance of the combined effect of HPV and ErbB-2 in HN cancer.Earlier stud...