The chimeric Ad5T/41sSK fiber represents a promising novel scaffold for peptide ligand insertion, especially considering its reported reduced biodistribution to the liver after2 injection. However, its suitability for applications in the context of oncolytic Ads, which depend on full replication potency, remains to be demonstrated. For specific applications requiring tumor-specific activity but not liver-de targeting, we considered the CAR binding-ablated HAdV-5 fiber as candidate scaffold suitable for ligand insertion. We show in this study that functional YSA peptide Sodium ascorbate insertion is feasible in the CD, FG and HI loops with the HI loop giving the best results. However, the CD and FG insertion sites might still be suitable for other peptide ligands, since our data for the chimeric Ad5T/41sSK fiber show that insertion site preferences depend on the inserted peptide. Several previous studies showed functional peptide ligand insertion into the HI loop or fusion to the C-terminus of the HAdV-5 fiber. Moreover, Ads with RGD peptide insertion into the HI loop, featuring enhanced infectivity, are being investigated in clinical gene therapy and virotherapy studies. Internal insertion sites other than the HI loop have been rarely studied, but should be considered in the future. Transduction by Ad5KO-HI-YSA was slightly more effective and selective compared with Ad5T/41sSKIJ-YSA in vitro. Thus, both virus formats warrant further investigation in gene therapy and virotherapy applications. In xenograft experiments, we found that i.t. virus injection resulted in strongly reduced tumor transduction for Ad5T/41sSK when compared with Ad5WT, while this was not the case for Ad5KO1. Of note, this was observed also for xenografts of CAR negative C8161 cells. These results point at CAR Siramesine independent tumor transduction in vivo mediated by HAdV-5 fibers, a conclusion also supported by a previous study reporting that CAR binding ablation does not de-target HAdV-5 vectors after i.t. injection. Thus, we show that Ad5T/41sSK possesses superior de-targeting features after i.t. application, while CAR binding-ablated HAdV-5 fibers require further optimization for efficient de-targeting.