Adenoviruses are used for cancer gene therapy as vectors for introducing the foreign genetic material to the tumors or cells that support tumor growth such as endothelial cells undergoing angiogenesis. Incorporation of targeting motif into the capsid protein of replication deficient adenovirus can increase the specificity of binding of the adenoviral vector on the target cell. Targeting motif NGR binds to aminopeptidase N which is highly expressed in endothelial cells undergoing angiogenesis, thereby represents a possible target for retargeting the adenoviral vector containing NGR peptide motif with the aim of inhibiting angiogenesis. It was shown that the amino acid environment has an influence in the specificity of retargeting adenoviral vectors, and that cyclization mediated by disulfide bonds has an important role. The aim of this work was to investigate the existence of disulfide bonds in retargeting motif C539NGRC543VSGC547AGRC551 inserted in HI loop of the fiber AdFNGR and in case of their existence, identify by mass spectrometry which cysteines are involved in disulfide bond formation. Further, the aim was to investigate the significance of disulfide bond formation on retargeting and transduction efficiency of rhabdomiosarcoma (RD) and breast cancer (MDA-MB-435S) cell lines with AdFNGR pretreated with dithiothreitol. Mass spectrometry has shown that cystein at position 539 does not form disulfide bond with any of the three cysteines in retargeting motif C539NGRC543VSGC547AGRC551, but this experimental approach did not prove whether there is a disulfide bond between cysteines C543, C547 and C551. Treatment of AdFNGR by dithiothreitol abolish increased transduction efficiency of RD and MDA-MB-435S cell lines as compared to Adwt, suggesting a possible existence of disulfide bonds in retargeting motif which allows a more favourable conformation of HI loop in AdFNGR and consequently better binding and internalization of adenovirus in the cell.