Abstract Background Delivery of transgenes into specific tissues by adenovirus vectors (AdVs) relies on ablations of their natural tropism and on introduction of a new tropism. If the interaction with its natural receptor is ablated, a new packaging cell line is required to produce the AdV. In the present study, we have used two de novo designed peptides (E‐Coil and K‐Coil) that interact with each other with high affinity to establish a new receptor‐ligand system for the propagation of retargeted AdVs. Methods We produced a cell line (293E) expressing on its surface a pseudoreceptor containing the E‐Coil. An AdV (AdFK4m/GFP) lacking the interaction with the primary receptor for adenovirus (CAR) and containing the K‐Coil inserted at the fiber C‐terminus was constructed and tested using two strategies: (1) an RGD motif (Arg‐Gly‐Asp) was inserted into the HI‐loop of the fiber; (2) AdFK4m/GFP was conjugated to a bispecific adaptor for the epidermal growth factor receptor (EGFR). Results AdFK4m/GFP infected 293E cells more efficiently than cells lacking the pseudoreceptor. The transduction was due to the K‐Coil/E‐Coil specific interaction since it was competed by addition of soluble K‐Coil, but not soluble fiber. We demonstrated that the modified AdV was retargeted toward αv integrin by inclusion of the RGD motif, or toward EGFR using the bispecific adaptor. Conclusions We have established a new system to produce AdVs ablated of natural tropism. This system should permit the retargeting of AdVs by inserting new ligands within the fiber or through the interaction with bispecific adaptors. Copyright © 2008 John Wiley & Sons, Ltd.