Even though adenoviral vector is widely used in gene therapy of squamous cell carcinoma of the head and neck (SCCHN), the expression level of Coxsackievirus and adenovirus receptor (CAR) in SCCNH is not clearly defined. To identify this variability, the expression of CAR was measured using SCCHN cell lines and compared with transfection efficiency. It was found by RT-PCR and Western blot analysis that CAR levels varied in SCCHN cell lines. FACS analysis and adenovirus infection assay revealed that there was a good correlation between the level of CAR expression and the transfection efficiency. To identify the actual CAR expression patterns of human SCCHN tissues in vivo, immunohistochemical staining was undertaken on frozen biopsies of six SCCHN patients. In all the patients examined, the normal tissues showed much stronger staining for CAR than the tumor tissues. These results demonstrate that the level of CAR expression of a tumor should be evaluated before clinical application of adenoviral vector for gene therapy in SCCHN.