Adenovirus-mediated gene transfer is a novel treatment strategy for head and neck squamous cell carcinoma (HNSCC) that may improve the unacceptable morbidity and mortality associated with conventional treatment. Efficient adenoviral (AdV) infection largely depends on cellular expression of the human coxsackie and adenovirus receptor (hCAR); however, the relatively recent identification of this receptor precludes a comprehensive description of its tissue distribution. We have created tissue culture model systems that approximate the differentiation and three-dimensional structure of stratified squamous epithelium characteristic of head and neck mucosa. Using these systems, we have found that expression of hCAR in native and modeled normal oropharyngeal epithelium decreased as cells differentiated with the most superficial and differentiated cells expressing no detectable hCAR. In contrast, modeled stratified HNSCC cells, which did not differentiate morphologically and did not express cytokeratin markers of differentiation, had equivalent expression of hCAR in superficial and basal layers. The expression of hCAR in our models correlated not only with the undifferentiated state, but also with efficiency of AdV infection. Despite expression of hCAR in underlying basal and suprabasal cells, topical application of AdV to normal modeled epithelium resulted in inefficient transduction of the most superficial cell layer without any infection of underlying cells. These data suggest that in normal epithelium the overlying squamous cells act as a barrier preventing infection of underlying cells that would otherwise be easily infected. In modeled stratified HNSCC, transduction was much more efficient and occurred up to four cell layers deep, suggesting that unlike normal superficial epithelial cells, the superficial cells of stratified HNSCC do not act as an effective barrier to adenoviral infection. The distribution of hCAR in native tissue and the enhanced susceptibility of undifferentiated oropharyngeal epithelial cells, including undifferentiated cancer cells, to AdV infection has important implications for the development of AdV-based targeting strategies for the treatment of head and neck cancer or premalignancies.