Targeting adenovirus encoding therapeutic genes to specific cell types has become a major goal in gene therapy. Coxsackievirus and adenovirus receptor (CAR) and α_V integrins have been identified as the primary cell surface components that interact with adenovirus type 5 (Ad5)-based vectors during in vitro transduction. Redirecting Ad5-based vectors requires abrogation of the natural interaction between the viral capsid and its cellular receptors and simultaneous introduction of a new binding specificity into the viral capsid. To abrogate native Ad5 tropism, fiber knob mutations Pro409Glu and Lys417Ala were each incorporated into adenoviral vectors, while the RGD motif was deleted from the penton base. In vitro transduction experiments showed that these capsid mutations eliminated Ad5 interactions with CAR and α_V integrins. Moreover, incorporation in the fiber HI loop of a vitronectin-derived ligand (VN4) specific for the uPAR/CD87 receptor provided the Lys417Ala virus with an alternative entry pathway specific for uPAR-expressing cells, indicating a successful in vitro retargeting of the vector. Unexpectedly, however, simultaneous disruption of Ad5 binding to CAR and α_V integrins had no effect on liver gene transfer following systemic administration in mice. This study highlights the need to understand better the molecular determinants involved in adenovirus uptake by the liver to control the fate of adenoviral vectors in vivo.