Interactions of the integrins alpha(4)beta(7) with its cognate ligand mucosal addressin cell adhesion molecule-1 (MAdCAM-1) play a crucial role in the development of mucosa-associated lymphoid organs, in the generation of mucosal immune responses, and in diverse pathological processes such as chronic inflammatory bowel disease and type I diabetes. Using a previously developed spatial screening technique we describe the development of potent and selective alpha(4)beta(7) integrin antagonists based on the domain 1 Leu-Asp-Thr (LDT) sequence of MAdCAM-1 that is essential for alpha(4)beta(7) integrin binding. A library of homodetic cyclic penta- and hexapeptides was synthesized presenting the pharmacophoric LDT-sequence in different conformations. The cyclic hexapeptide P10 cyclo(Leu-Asp-Thr-Ala-D-Pro-Ala) inhibits alpha(4)beta(7) integrin mediated cell adhesion to MAdCAM-1 effectively. Further optimization of the lead structure P10 resulted in cyclic hexapeptides with enhanced activity. The compounds P25 cyclo(Leu-Asp-Thr-Ala-D-Pro-Phe), P28 cyclo(Leu-Asp-Thr-Asp-D-Pro-Phe), P29 cyclo(Leu-Asp-Thr-Asp-D-Pro-His), and P30 cyclo(Leu-Asp-Thr-Asp-D-Pro-Tyr) strongly inhibited alpha(4)beta(7) integrin mediated cell adhesion to MAdCAM-1, but they did not affect binding of the closely related alpha(4)beta(1) integrin to VCAM-1.