In an effort to improve the therapeutic outcome for squamous cell cancer of the head and neck, we have used the enzyme cytosine deaminase (CD) and the prodrug 5-fluorocytosine (5-FC) as a means to deliver the chemotherapeutic agent 5-fluorouracil (5-FU) in a tumor-specific manner and have evaluated the use of this treatment in combination with external-beam radiation. Infection of SCCVII cells in culture with a CD-expressing retrovirus and treatment with 5-FC was cytotoxic depending on the time of treatment and dose of 5-FC. An orthotopic model of squamous cell cancer of the head and neck was used in vivo to study the CD/5-FC system both alone and with concurrent radiation due to the radiosensitizing properties that 5-FU generates in situ. Treated mice were imaged using magnetic resonance imaging (MRI), and their survival was evaluated. Neither 5-FU nor radiation either alone or combined provided a survival advantage. In contrast, 5-FC treatment prolonged survival and decreased tumor burden compared to control animals, but the tumors recurred after the treatment ceased. Finally, combined treatment with concurrent administration of 5-FC and radiation resulted in a synergistic decrease in tumor growth and enhanced survival over treatment with 5-FC or radiation alone.