In this study, a prototype Adenovirus type 5 (Ad5) vector deleted of the fiber knob domain and carrying an Affibody molecule as the targeting ligand showed decreased susceptibility to human pre-existing antibodies. This vector, Ad5/R7-Z(taq)Z(taq), has short fibers carrying seven shaft repeats, a non-native trimerization signal and an affibody molecule (Z(taq)) reactive to Taq polymerase. Ad5/R7-Z(taq)Z(taq) could be specifically targeted to 293 cells stably expressing membrane-bound anti-Z(taq) idiotypic affibody called Z(ztaq) (293Z(ztaq)). Sera from 50 blood donors were analyzed for neutralization activity (NA) against the parental Ad5/Fiwt vector and knobless Ad5/R7-Z(taq)Z(taq) on 293Z(ztaq) cells. Twenty-three sera had NA titers (>= 1:64) against Ad5/Fiwt (46%) and only two against Ad5/R7-Z(taq)Z(taq) (4%). Characterization of sera with NA titers showed that the knob domain is one of the targets of the antibodies. Neutralization assays using sera pre-adsorbed on knob and hexon proteins showed that the NA of the sera was carried mainly by anti-knob and anti-hexon antibodies, but in certain sera the anti-hexon antibodies represent the major population of the neutralizing antibodies (NAbs). Our results suggested that a combination of knob deletion and hexon switching could be an effective strategy for Ad vectors to better evade the anti-Ad NAbs.