Glucagon-like peptide-2 (GLP-2) is a 33 amino acid gastrointestinal hormone that regulates epithelial growth in the intestine. Dipeptidylpeptidase IV cleaves GLP-2 at the position 2 alanine, resulting in the inactivation of peptide activity. To understand the structural basis for GLP-2 action, we studied receptor binding and activation for 56 GLP-2 analogues with either position 2 substitutions or alanine replacements along the length of the peptide. The majority of position 2 substitutions exhibited normal to enhanced GLP-2 receptor (GLP-2R) binding; in contrast, position 2 substitutions were less well tolerated in studies of receptor activation as only Gly, Ile, Pro, alpha-aminobutyric acid, D-Ala, or nor-Val substitutions exhibited enhanced GLP-2R activation. In contrast, alanine replacement at positions 5,6,17, 20, 22, 23, 25, 26, 30, and 31 led to diminished GLP-2R binding. Position 2 substitutions containing Asp, Leu, Lys, Met, Phe, Trp, and Tyr, and Ala substitutions at positions 12 and 21 exhibited normal to enhanced GLP-2R binding but greater than 75% reduction in receptor activation. D-Ala(2), Pro(2) and Gly(2), Ala(16) exhibited significantly lower EC(50)s for receptor activation than the parent peptide (p < 0.01-0.001). Circular dichroism analysis indicated that the enhanced activity of these GLP-2 analogues was independent of the alpha-helical content of the peptide. These results indicate that single amino acid substitutions within GLP-2 can confer structural changes to the ligand-receptor interface, allowing the identification of residues important for GLP-2R binding and receptor activation.