Recombinant adenoviral vectors are attractive in the context of cancer gene therapy because they are capable of delivering genes to a wide variety of tissues. The utility of adenoviruses is limited by their lack of specificity and by the absence of the receptor(s) for these viruses on many tumor cells. Redirecting adenoviral vectors to tissue- or tumor-specific targets can be achieved by using bispecific conjugates produced by chemical linkage of an anti-adenovirus antibody (Ab) and a ligand or Ab directed toward a specific target. To avoid the limitations of chemical conjugates, molecular conjugates of anti-fiber knob and ligand have been proposed. We present here a novel strategy that allows the production of recombinant bispecific single-chain Abs directed at cell surface molecules. A construct was made that encodes a neutralizing anti-adenovirus fiber single-chain Fv (scFv) Ab (S11) fused to a scFv Ab (425) directed against the epidermal growth factor receptor. The fusion protein markedly enhanced the infection efficiency of adenoviral vectors in epidermal growth factor receptor-expressing cell lines. The bispecific scFv could be purified and concentrated after binding of its 6His tag to a nickel column without significant loss of activity. This approach should permit the production of high quantities of active bispecific scFv for in vivo use. The universal design of the construct allows rapid screening for relevant specific scFv directed at cell surface antigens that can be incorporated into adenoviral targeting strategies.