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Purpose Imaging of glioblastoma multiform (GBM) tumor using 68-Galium-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid-Ser-Ser-Ser-Leu-Thr-Val-Ser-Pro-Trp-Tyr (68Ga-DOTA-(Ser)3-LTVSPWY) as a PET radiotracer for HER2 receptor due to fact that this receptor plays a pivotal role in the tumorigenesis and tumor progression in a wide range of cancer. Methods 68Ga-DOTA-(Ser)3-LTVSPWY was produced with high radiochemical purity. The affinity and specificity of this radiotracer toward HER2 receptor on the surface of glioma glioblastoma (U-87 MG) cell line were evaluated. Furthermore, the biodistribution and PET imaging of this radiolabeled peptide were investigated on xenografted U-87 MG tumor-bearing mice. Results The in-vitro specific binding study revealed that the 68Ga-DOTA-(Ser)3-LTVSPWY binds to different cell lines with respect to their level of HER2 expression. The calculated KD and Bmax of radiolabeled peptide toward U-87 MG cell line were 5.5 ± 2.4 nmol/l and (2.4 ± 0.3) × 105 receptors per cell, respectively. The highest tumor uptake was observed at 30-min postinjection, whereas the tumor-to-muscle ratio was about four-fold. The acquired PET images distinctively show tumor site, which was blocked with excess nonlabeled peptide that revealed specific in-vivo targeting of 68Ga-DOTA-(Ser)3-LTVSPWY for glioma. Conclusion 68Ga-DOTA-(Ser)3-LTVSPWY specifically recognizes HER2 receptors and could be a potential candidate for GBM imaging.