Conventional cancer treatments are not adequate for the majority of most patients stricken with squamous cell carcinomas of the head and neck (SCCHN). Conditionally replicating adenoviruses (CRAds) represent a promising new modality for treating of neoplastic diseases, including SCCHN. Specifically, CRAd agents infect tumor cells and selectively replicate within them, thus causing their death while sparing surrounding normal cells in the host. Oncolysis results from the replicative life cycle of the virus, which lyses infected tumor cells and releases viral progeny for propagation of infection and resultant lysis of neighboring cancer cells, sparing normal host cells. However, to date there have been two main limitations to successful clinical application of these CRAd agents: poor infectivity and poor tumor specificity. Here we report the construction of a CRAd agent, CRAd-CXCR4.F5/3, in which the adenovirus E1 gene is driven by a tumor-specific CXCR4 promoter, and the viral infectivity is enhanced by a fiber modification, F5/3, containing an Ad3 knob chimeric fiber protein. As expected, this agent improved both of the viral infectivity and tumor specificity as evaluated in established SCCHN tumor cell lines and in primary tumor tissues from multiple patients. As an added benefit, the activity of the CXCR4 promoter was low in human liver as described previously. Based on these data, the CRAd-CXCR4.F5/3 is a promising novel CRAd agent for SCCHN targeting with low host toxicity.