We investigated entry targeting of Ads by genetic insertion of a targeting peptide into chimeric fibers with HAdV-41 knob as a de-targeted scaffold. To this end, we inserted the 12-mer EphA2binding peptide YSA flanked by short linkers into the HI, IJ or EG loops of this knob domain. To explore the relevance of shaft length on YSA-mediated Ad transduction, we combined these YSA-containing knobs with the short HAdV-41 fiber shaft or the long HAdV-5 fiber shaft. In a third set of fibers, we incorporated the long HAdV-5 fiber shaft with a mutated heparin sulfate proteoglycan -binding motif. This mutation was reported to confer improved de-targeting. After plasmid transfection, all constructs were expressed and Moclobemide possessed trimerization capacity, but trimerization was clearly less efficient for the long-shafted constructs, especially those containing the peptide in the HI loop. Using a combined transfection/superinfection protocol, we were able to produce high titer pseudo typed LacZ reporter Ad vectors with all fiber formats. Incorporation of fiber molecules into viral particles was efficient for the short-shafted fiber and, despite reduced trimerization capacity, for the long shafted IJ-YSA fibers. Long-shafted EG-YSA and HIYSA fibers showed reduced or lacked fiber incorporation into virus particles, respectively. Next, we analyzed EphA2 Triclabendazole expression in a panel of pancreatic cancer cells, melanoma cells, endothelial cells, and a hepatic cell line. We detected strong EphA2 expression for pancreatic cancer cells, endothelial cells, and for 4 of 6 melanoma cell cultures. EphA2 was not detected for the two melanoma cell lines SKMEL-28 and Mel624 and the hepatic cell line HepG2. Transduction experiments with EphA2-positive cell lines PANC-1, MIA PaCa-2, Capan-1 and C8161 yielded results that were similar for the respective cell lines: All three viruses pseudo typed with the short-shafted YSA fibers showed transduction, which was substantially stronger than for the control viruses without YSA peptide. For viruses with unmodified HAdV-5 shaft, the insertion of YSA into the HI, IJ and EG loops showed weak, strong, and intermediate transduction efficiency, respectively. The long-shafted IJ-YSA virus was even superior to the short shafted viruses.