Currently available therapeutic modalities for advanced head and neck squamous cell carcinoma (HNSCC), such as radical surgery, high-dose radiotherapy, and chemotherapy, are of limited efficacy. Overall survival has not significantly improved over the past 30 years; thus, HNSCC represents a promising target for new therapeutic approaches, such as gene therapy. A major obstacle to the development of effective suicide gene therapy strategies that rely on in situ transduction of tumor cells is the poor distribution of the vector throughout the tumor. To address this problem we evaluated the use of Ad.OW34, an Elb 55 kD and HSV-tk-carrying, replication-competent adenoviral vector that has a wild-type adenovirus phenotype in replicating cells, in combination with ganciclovir (GCV) as a treatment for HNSCC xenografts in nude mice and compared its efficacy with that of a standard replication-deficient adenovirus expressing HSV-tk (Ad.TK). In this model, Ad.OW34 had a significantly greater antitumor effect than the traditional Ad.TK vector, administered alone or in combination with GCV. Interestingly, GCV did not further enhance the oncolytic efficacy of Ad.OW34. GCV also aborts viral replication and thus represents a fail-safe feature of this vector not found in wild-type adenovirus.