Transduction of malignant glioma with adenovirus serotype 5 (Ad5) vectors is limited by the low levels of coxsackievirus and adenovirus receptor (CAR) on tumor cells. However, malignant brain tumors have been found to overexpress a glioma-associated receptor, inlerleukin-13 receptor α2 chain (IL-13Rα2), a marker of both glial transformation and tumor grade. To selectively target Ad5 to IL-13Rα2, we constructed a replication-deficient adenoviral vector that possesses an IL-13 ligand presented by a T4 phage fibritin shaft, and designated the new virus LU-13. Western blot and sequence analyses confirmed proper trimerization and ligand presentation by the T4 fibritin shaft. Confocal microscopy analysis of primary glioma suspensions incubated with viral recombinants showed that LU-13 colocalized with IL-13Rα2. Luciferase transduction assays conducted in both primary and passaged glioma cell cultures exhibited at least 10-fold enhanced gene transduction. Moreover, the virus preferentially bound to glioma cells, as documented by increased adenoviral E4 DNA copy number. In vitro competition assays performed with anti-human IL-13 monoclonal antibody confirmed significant attenuation of LU-13 transduction. These results were further confirmed in vivo, where LU-13 showed a 300-fold increase in transgene expression. In summary, we describe here the development of a novel and targeted adenoviral vector that binds IL-13Rα2. Our findings confirm the ability of LU-13 to bind IL-13Rα2 and increase transgene expression, making it an attractive gene therapy vector for the treatment of malignant glioma in a clinical setting. © Mary Ann Liebert, Inc.