To improve safety and specificity of oncolytic adenoviruses, we introduced T-cell receptors (TCR) specific for a unique class of truly tumor-specific antigens into the adenoviral fiber protein. The adenoviral fiber knob responsible for attachment to the coxsackie-adenoviral receptor (CAR) on target cells was replaced by a single-chain TCR (scTCR) molecule with specificity for the melanoma-associated cancer-testis antigen MAGE-A1, presented by HLA-A1, and an extrinsic trimerization motif in a replicating Ad5 vector (Ad5.R1-scTCR). The production of the recombinant virus was initiated in a novel producer cell line that expressed an antibody-based hexon-specific receptor (293T-AdR) in the cell membrane. This new production system allowed CAR-independent and target antigen-independent propagation of Ad5.R1-scTCR. Infection with adenovirus bearing the scTCR-based fiber resulted in an efficient killing of target tumor cells. The infection was cell type specific because only HLA-A1(+)/MAGE-A1(+) melanoma cells were killed, and thus, this retargeting strategy provides a versatile tool for future clinical application.