BACKGROUND: Peptides radiolabeled with fluorine-18 are frequently synthesized using prosthetic groups. Among them, activated esters of 6-[(18)F]fluoronicotinic acid ([(18)F]FNA) have been prepared and successfully employed for (18)F-labeling of diverse biomolecules, including peptides. The utility of [(18)F]FNA as a prosthetic compound has been demonstrated in both preclinical and clinical settings, including radiopharmaceuticals targeting prostate-specific membrane antigen and poly(ADP ribose) polymerase inhibitors. This study aims to evaluate a [(18)F]FNA-conjugated nonapeptide, [(18)F]FNA-N-CooP, for positron emission tomography imaging of intracranial BT12 glioblastoma xenografts in a mouse model. Additionally, this study highlights the importance of including control experiments with prosthetic compound alone when it constitutes a major radiometabolite. RESULTS: [(18)F]FNA-N-CooP successfully delineated intracranial glioblastoma xenografts yielding a standardized uptake value of 0.21 ± 0.03 (n = 4) and a tumor-to-brain ratio of 1.84 ± 0.29. Ex vivo autoradiography of tumor tissue showed a partial co-localization between radioactivity uptake and the target fatty acid binding protein 3 expression. However, in vivo instability of [(18)F]FNA-N-CooP was observed, with [(18)F]FNA identified as a major radiometabolite. Notably, control studies using [(18)F]FNA alone also visualized tumors, producing a standardized uptake value of 0.90 ± 0.10 (n = 4) and a tumor-to-brain ratio of 1.51 ± 0.08. CONCLUSIONS: Both [(18)F]FNA-N-CooP and [(18)F]FNA enabled PET visualization of human glioblastoma in the mouse model. However, the prominent presence of [(18)F]FNA as radiometabolite complicates the interpretation of [(18)F]FNA-N-CooP PET data, suggesting that the observed radioactivity uptake may primarily originate from [(18)F]FNA and other radiometabolites. Enhancing peptide stability is essential for improving imaging specificity. This study underscores the critical need to assess the imaging contributions of prosthetic groups when they function as significant radiometabolites.