Expression of cellular receptors determines viral tropism and limits gene delivery by viral vectors. Protein transduction domains (PTDs) have been shown to deliver proteins, antisense oligonucleotides, liposomes, or plasmid DNA into cells. In our study, we investigated the role of several PTD motifs in adenoviral infection. When physiologically expressed, a PTD from human immunodeficiency virus transactivator of transcription (Tat) did not improve adenoviral infection. We therefore fused PTDs to the ectodomain of the coxsackievirus-adenovirus receptor (CAR(ex)) to attach PTDs to adenoviral fiber knobs. CAR(ex)-Tat and CAR(ex)-VP22 allowed efficient adenoviral infection in nonpermissive cells and significantly improved viral uptake rates in permissive cells. Dose-dependent competition of CAR(ex)-PTD-mediated infection using CAR(ex) and inhibition experiments with heparin showed that binding of CAR(ex)-PTD to both adenoviral fiber and cellular glycosaminoglycans is essential for the improvement of infection. CAR(ex)-PTD-treated adenoviruses retained their properties after density gradient ultracentrifugation, indicating stable binding of CAR(ex)-PTD to adenoviral particles. Consequently, the mechanism of CAR(ex)-PTD-mediated infection involves coating of the viral fiber knobs by CAR(ex)-PTD, rather than placement of CAR(ex) domains on cell surfaces. Expression of CAR(ex)-PTDs led to enhanced lysis of permissive and nonpermissive tumor cells by replicating adenoviruses, indicating that CAR(ex)-PTDs are valuable tools to improve the efficacy of oncolytic therapy. Together, our study shows that CAR(ex)-PTDs facilitate gene transfer in nonpermissive cells and improve viral uptake at reduced titers and infection times. The data suggest that PTDs fused to virus binding receptors may be a valuable tool to overcome natural tropism of vectors and could be of great interest for gene therapeutic approaches.