Replication deficient adenoviruses type 5 (Ad5) are widely used vectors for cancer gene therapy. The retargeting of Ad5 is achieved by incorporation of a targeting motifs into the adenovirus capsid proteins, mostly in the HI-loop of fiber or hexon hypervariable region 5 (HVR5). Increased expression of αv integrins has been frequently shown in tumor cells as compared to normal cells. The targeting motif within Ad5 that retargets its transduction to cells expressing αv integrins is RGD. One of the strategies of tumor gene therapy is inhibition of angiogenesis. Endothelial cells in angiogenesis express aminopeptidase N (APN) which binds targeting motifs containing NGR. It is known that different amino acid environment and/or cysteine residues flanking targeting peptides RGD and NGR influence the affinity and specificy of binding to corresponding receptors. The aim of this work was to investigate the existance of disulfide bond/s in two replication deficient Ad5 vectors: (i) Ad5HCRGDC containing CRGDC retargeting motif inserted in HVR5 and (ii) Ad5ARAP containing CNGRCVSGCAGRC retargeting motif inserted in HI loop of the fiber protein. It was shown that treatment with dithiothreitol had no effect on Ad5HCRGDC transduction efficacy of human embryonal rhabdomyosarcoma (RD) cell line i.e. it did not influence binding to αv integrins. The proof that cysteines in the CRGDC motif do not form disulfide bond was given by mass spectrometry. Treatment of Ad5ARAP by ditiothreitol abolished retargeting to APN that is expressed in RD cell line. Therefore, cysteines surrounding NGR retargeting motif within CNGRCVSGCAGRC form disulfide bond/s crucial for APN binding. The analysis of amino acid residues surrounding retargeting motifs enables better understanding of the relation between their structure and function which helps in design of improved retargeting adenoviruses for tumor gene therapy.