Viruses are commonly investigated as vector systems for gene therapy. To be effective, virus-mediated gene-delivery systems require the presence of specific virus receptors to enter the target cell. One example is adenovirus and its primary receptor is the coxsackievirus and adenovirus receptor (CAR). Madin-Darby canine kidney (MDCK) cells have become a choice model system for studying CAR and adenovirus infection due to their ability to polarize rapidly into an epithelium with high transepithelial resistance. We show here that, whilst MDCK cells are resistant to adenovirus infection and hence appear functionally CAR-deficient, polarized MDCK cells express significant levels of CAR sequestered on the basolateral surface, where it is inaccessible for virus infection. Thus, although a cell type may be resistant to adenovirus infection, it is impossible to know whether it is due to a deficiency, as both CAR absence and inaccessibility are barriers to adenovirus-mediated gene transfer.