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(-/-)) 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(-/-) 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.