Purpose <p>Transferrin receptor 1 (TfR1) is a transmembrane glycoprotein that mediates cellular iron uptake. Despite its potential as a target for tumor diagnosis, the lack of suitable TfR1 ligands has hindered the development of effective TfR1 imaging agents. We labeled a recently discovered TfR1 ligand, TfRB1G3, with Ga-68 and evaluated its potential for TfR1 imaging through preclinical experiments.</p> Methods <p>TfRB1G3 was modified with HBED-CC and radiolabeled with ^67/68Ga ([^67/68Ga]Ga-TfRB1G3). Human glioblastoma (U87MG) cells were transfected with TfR1-targeting siRNA (TfR1-knockdown) or non-targeting siRNA (control), and accumulation of [⁶⁷Ga]Ga-TfRB1G3 was evaluated. The K_D value of [⁶⁷Ga]Ga-TfRB1G3 for TfR1 was also calculated. Ex vivo biodistribution was conducted in normal and U87MG tumor-bearing mice after injecting [⁶⁷Ga]Ga-TfRB1G3. PET imaging studies were conducted after injecting [⁶⁸Ga]Ga-TfRB1G3, with/without co-injection of 10 nmol TfRB1G3 in mice bearing U87MG tumors.</p> Results <p>[^67/68Ga]Ga-TfRB1G3 was obtained with high radiochemical purity (&gt; 90%). The accumulation of [⁶⁷Ga]Ga-TfRB1G3 in the TfR1-knockdown cells (22.0 ± 1.1% Dose/mg) was significantly lower than that in the control (308.5 ± 14.3% Dose/mg). The K_D value was 1.0 nM. Tumor accumulation of [⁶⁷Ga]Ga-TfRB1G3 was higher than in any other organs except the kidneys, and co-injection of TfRB1G3 significantly reduced the tumor accumulation by more than 90%. PET imaging with [⁶⁸Ga]Ga-TfRB1G3 clearly visualized TfR1-positive tumor as early as 20&#xa0;min post-injection.</p> Conclusion <p>[⁶⁸Ga]Ga-TfRB1G3 demonstrated excellent affinity and specificity for TfR1 both in vitro and in vivo, providing clear PET images of TfR1-positive tumors shortly after injection. [⁶⁸Ga]Ga-TfRB1G3 would enable sensitive and quantitative imaging of TfR1 expression.</p>