Telomerase-specific replication-competent adenovirus, Telomelysin (OBP-301), has a human telomerase reverse transcriptase promoter that regulates viral replication and efficiently kills human cancer cells. The objectives of this study are to examine the effects of OBP-301 in squamous cell carcinoma of the head and neck cells in vitro and in xenografted animals in vivo. OBP-301 was found to be cytotoxic to the YCUT892, KCCT873, KCCT891, KCCL871, YCUM862, HN12, and KCCOR891 cell lines in vitro. However, the level of cytotoxicity is not correlated with the expression levels of adenoviral receptors, which may be required for adenoviral infection in squamous cell carcinoma of the head and neck cells. OBP-301 shows remarkable antitumor activity against established s.c. KCCT873 tumors in immunodeficient animals in a dose-dependent manner. In addition, no significant toxicity was observed in animals receiving treatment. These results suggest that OBP-301 is a novel therapeutic agent with promise for the treatment of human head and neck cancers.