Systemic administration of adenoviral vectors leads to a widespread distribution of vector. Therefore, targeting of adenoviral vectors to specific tissues or cell types will require methods to ablate the normal tropism of the vector simultaneously with the introduction of new receptor specificities. To inhibit native receptor binding, we mutated residues in the AB loop of the adenovirus type 5 (Ad5) fiber. We genetically incorporated the S408E-P409A mutation, referred to as KO1, into the adenoviral genome alone or in combination with an RGD-targeting ligand in the HI loop of fiber. Transduction experiments confirmed that the KO1 mutation results in a significant reduction in fiber-dependent gene transfer on A549 and primary fibroblast cells that could be restored via the RGD-targeting ligand. Competition transduction experiments verified the receptor-binding properties of each vector on A549 and hepatocytes in vitro. Unexpectedly, in mice systemic delivery of the vector containing the KO1 mutation resulted in efficient liver transduction that was localized specifically to hepatocytes. We confirmed these results in three different mouse strains, indicating that hepatic adenoviral gene transfer may be independent of the coxsackievirus–adenovirus receptor and that in vivo retargeting will require further viral capsid modifications to generate a fully detargeted adenoviral vector upon which to introduce new tropisms.