Gene therapy vectors based on human adenovirus (Ad) serotypes 2 and 5 continue to show increasing promise as gene therapy delivery vehicles. Early gene therapy efforts, however, revealed that the clinical benefit of these gene delivery modalities was irrevocably linked to specific localization of the therapeutic agent. Hence, a fundamental requirement for somatic cell gene therapy is targetable gene delivery vectors. To this end, Ads incorporating genetic modifications have provided gains in vector efficiency; however, specific vector targeting to specific cell-surface molecules is still required. For cell-specific targeting, the use of antibodies or single chain antibody fragments (scFv) remains a very attractive concept made possible by the discovery of disease-specific membrane antigens and a wide selection of cognate antibody molecules. However, a barrier to the development of Ad vectors with genetically incorporated scFvs occurs when the antibody requires posttranslational modifications not available to Ad capsid proteins due to their cytoplasmic routing during virion assembly. To overcome this problem, we have developed a targeting strategy that uses a secreted scFv that undergoes the requisite posttranslational modification (disulfide bond formation) in the endoplasmic reticulum and is trafficked for secretion. Formation of the vector/ligand targeting complex occurs following cell lysis and is achieved via genetic tagging of the Ad capsid and scFv with synthetic leucine zipper-like dimerization domains that have been optimized for structural and biological compatibility with the Ad capsid and the secreted scFv. Importantly, we have designed our Zipper domains in pairs, such that only compatible heterodimers can associate, thus eliminating the possibility of aggregate formation. Zipper domains were incorporated into the C-terminus of Ad5 fiber and our knob-deleted chimeric fiber molecule, fiber-fibritin (FF). The ligand is comprised of a scFv fused with the kappa-chain IgG signal peptide and a Zipper peptide. Our results show that Zipper domain-containing fiber or FF molecules trimerize and associate with the virion penton base protein and that these vectors can be amplified. Further, we demonstrate that peptide Zippers can be genetically fused to scFv without decreasing ligand recognition of antigen. Most important, we show that Zipper-tagged virions and scFv can self-associate and form targeting complexes capable of binding to target receptors. This approach to targeting ligand design solves the problem of structural and biosynthetic compatibility with the Ad and thus facilitates targeting of the vector to a variety of cellular receptors. Since this gene transfer is independent of the expression of the native Ad5 receptor by the target cells, this strategy results in the derivation of truly targeted Ad vectors suitable for tissue-specific gene therapy.