E2F-1, a transcription factor by discovery, is thought to play a crucial role in regulating G1/S cell cycle progression. Its activity is modulated by complex formation with the retinoblastoma protein and related proteins. Overexpression of E2F-1 has been shown to induce apoptosis in quiescent fibroblasts. We constructed a recombinant E2F-1 adenovirus to test whether an overexpression of E2F-1 in head and neck squamous cell carcinoma cell lines would also induce apoptosis. Two cell lines, Tu-138 and Tu-167, were chosen for use in this study. Both cell lines harbor p53 mutations but express different levels of the retinoblastoma protein. Upon E2F-1 adenovirus infection, both cell lines expressed elevated levels of E2F-1 protein and then activated a pRb-chloramphenicol acetyltransferase reporter construct containing an E2F-1 binding motif. In vitro growth assay demonstrated that growth suppression by the E2F-1 protein was effective on both cell lines. Results from DNA fragmentation and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling analyses indicated apoptosis induction in cells infected with AdCMV-E2F-1. Moreover, ex vivo experiments in nude mice showed total suppression of tumor growth at sites that received cells infected AdCMV-E2F-1. An in vivo analysis of apoptosis using in situ end-labeling further demonstrated the induction of apoptosis by AdCMV-E2F-1 in tumor-bearing animals. These data indicate that overexpression of E2F-1 via an adenoviral vector suppresses in vitro and in vivo growth of head and neck squamous carcinoma cell lines through induction of apoptosis.