Purpose <p>Native ligand-derived peptides have significantly advanced the development of targeting ligands in radiopharmaceutical discovery and design. Herein, we report the first attempt to develop novel mesenchymal–epithelial transition factor (c-MET) targeted peptide positron emission tomography (PET) probes based on the endogenous biomolecule, hepatocyte growth factor (HGF).</p> Methods <p>Three c-MET targeted peptides were designed from the N-terminal and kringle 1 domain (NK1: 32–207 amino acid residues) of HGF. Then they were conjugated with chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and radiolabeled with [⁶⁸Ga]GaCl₃. The resulted [⁶⁸Ga]Ga-labelled probes, [⁶⁸Ga]Ga-DOTA-K1, [⁶⁸Ga]Ga-DOTA-A-C5, and [⁶⁸Ga]Ga-DOTA-A-M8 were evaluated in vitro and in vivo.</p> Results <p>Among three PET probes, [⁶⁸Ga]Ga-DOTA-A-M8 exhibited a high affinity for c-MET (IC₅₀ = 5.43 nM) and demonstrated specific and high uptake in c-MET highly expressing HCT-116 cells. Small animal PET/CT imaging clearly visualized the tumor with good contrast using [⁶⁸Ga]Ga-DOTA-A-M8 over 120&#xa0;min. Quantitative analysis of PET images revealed tumor uptake of [⁶⁸Ga]Ga-DOTA-A-M8 at 30&#xa0;min post-injection was 2.91 ± 0.20%ID/g in HCT-116 models. The probe was mainly cleared out through the kidney-bladder pathway. Biodistribution study showed HCT-116 tumor uptake of 2.84 ± 0.07%ID/g and 0.54 ± 0.09%ID/g in the normal and blocking mice group, respectively, at 30&#xa0;min post-injection. The tumor-to-muscle, tumor-to-blood and tumor-to-liver ratios were 3.60 ± 1.09, 1.48 ± 0.24 and 2.48 ± 0.28, respectively, at 30&#xa0;min.</p> Conclusion <p>A novel c-MET-targeting PET probe, [⁶⁸Ga]Ga-DOTA-A-M8, has been successfully developed in this study. It demonstrates high tumor-targeting capability and specificity. This study highlights developing PET probes based on native ligands is a powerful and generalizable strategy.</p> Graphical abstract <p></p>