PURPOSE: This study aimed to assess the dynamics of tumor-specific uptake, retention, and blood-tumor barrier penetration of our unique IL-13 receptor α-2 × CD3 bispecific T-cell engager (BTE) following systemic administration in mice with intracranial glioblastoma (GBM) xenografts. EXPERIMENTAL DESIGN: In vitro, BTE binding and accumulation were evaluated in glioma neurospheres. In in vivo studies, the BTEs labeled with either iodine-124 using residualizing chemistry or conjugated to Cy5 were used for longitudinal tracking in patient-derived xenograft models of GBM using PET/CT and confocal microscopy. The survival analysis in mice bearing intracranial GBM tumors was conducted to validate the findings from imaging studies. RESULTS: In vitro, the BTE demonstrated target-specific binding and accumulation in IL-13 receptor α-2-expressing glioma spheres. In vivo, PET/CT imaging revealed that the BTE reached the tumor site within 3 hours after injection, achieving up to 4.8% ID/g, with sustained tumor retention for up to 24 hours, significantly higher than background levels in surrounding normal brain tissue. Confocal microscopy confirmed BTE presence in the tumor bed extravascular space with evidence of T cell-mediated BTE transport across the blood-tumor barrier. Despite its short plasma half-life, the BTE remained in the tumor microenvironment for at least 24 hours. Mice bearing GBM6 brain tumors treated with BTE for 3 to 4 days apart via the intravenous route showed a significant survival advantage over the control group. CONCLUSIONS: Our findings provide critical insights into the pharmacokinetics of BTE molecules in GBM. They demonstrate effective penetration and prolonged intratumoral retention following a single systemic dose, supporting further exploration of BTE treatment regimens for translation to clinical settings.