Adenoviral vector systems for gene therapy can be much improved by targeting vectors to specific cell types. This requires both the complete ablation of native adenovirus tropism and the introduction of a novel binding affinity in the viral capsid. We reasoned that these requirements could be fulfilled by deleting the entire knob domain of the adenovirus fiber protein and replacing it with two distinct moieties that provide a trimerization function for the knobless fiber and specific binding to the target cell, respectively. To test this concept, we constructed adenoviral vectors carrying knobless fibers comprising the alpha-helix trimerization domain from MoMuLV envelope glycoprotein. Two mimic targeting ligands, a Myc-epitope and a 6His-tag, were attached via a flexible linker peptide. The targeted knobless fiber molecules were properly expressed and imported into the nucleus of adenovirus packaging cells, where they were incorporated as functional trimers into the adenovirus capsid. Both ligands were exposed on the surface of the virion and were available for specific binding to their target molecules. Moreover, the knobless fibers mediated gene delivery into cells displaying receptors for the coupled ligand. Hence, these knobless fibers are prototype substrates for versatile addition of targeting ligands to generate truly targeted adenoviruses.