Background: Glucagon-like peptide-2 (GLP-2) is a 33 amino acid pro-glucagon-derived hormone produced in the intestinal enteroendocrine L-cells with trophic actions on both the gut and bones. GLP-2(1-33) is cleaved by the ubiquitous protease dipeptidyl peptidase-4 (DPP-4), resulting in GLP-2(3-33) with competitive antagonistic properties on the GLP-2 receptor (GLP-2R). Here we present two new hGLP-2 radioligands with different pharmacodynamic profiles. Experimental Approach: The methionine in position 10 of GLP-2(1-33) was substituted with tyrosine to enable oxidative iodination with incorporation of the iodine isotope [125I]. Similar substitution was done in GLP-2(3-33), thereby creating two new radioligands; an agonist [125]-hGLP-2(1-33,M10Y) and an antagonist [125]-hGLP-2(3-33,M10Y). Both were characterized regarding competition binding, binding kinetics and target tissue autoradiography. Key results: High and similar binding affinities for the human GLP-2R were observed for [125I]-hGLP-2(1-33,M10Y) and [125I]-hGLP-2(3-33,M10Y) with KD values of 59.3 nM and 40.6 nM, respectively. The M10Y substitution did not change the functional properties of GLP-2(1-33) or GLP-2(3-33). The antagonist [125I]-hGLP-2(3-33,M10Y) had higher Bmax and faster on-rate for the hGLP-2R compared to the agonist [125I]-hGLP-2(1-33,M10Y). Using autoradiography in mice strong labeling was observed in subepithelial myofibroblasts (SEMF) and pancreas islet cells. Both radioligands were selective for the GLP-2R, except for a low affinity binding to the GLP-1R (IC50 of 130 and 330 nM, respectively) Conclusion and implications: We successfully developed two new high affinity radioligands for GLP-2R studies and identified SEMF and pancreatic islets as target for GLP-2. It is uncertain whether binding in the pancreas islets results from GLP-2R or GLP-1R binding.