The selective emigration of blood born leukocytes into tissues is mediated. in part by interactions of Ig-like cell adhesion molecules (IgCAMs) expressed on vascular endothelium and their cognate ligands. thc lcukocyte integrins. Within niucosal lymphoid tissues and gastrointestinal sites the inucosai vascular addressin. MAdCAM-I is the predominant IgCAM. mediating specific lymphocyte homing via interactions with its ligand on lymphocytes, the integrin 04/17. Previous studies have shown thal an essential binding motif resides in the first Ig domain of all IgCAMs, containing an acidic residue ( D or E) preceded by an aliphatic residue ( L or I ) that resides in strand C or the C D loop. However, domain swap experiments with MAdCAM-I and VCAM-I havc shown a requirement for both Igdoinains 1 and 2 for efficient integrin binding. Wedescribe the use of chimeric MAdCAM-I/VCAM-I receptors and point mutations in MAdCAM-I to define other sites that are required for binding t o the integrin (u4;j7. Wc find that, in addition to critical C D loop residues, other regions in both domain one and two contribute to MAdCAM-I /a407 interactions. including a buried arginine residue in the F strand of domain one and several acidic residues in a highly extended DE ribbon in domain 2. These mutations, when placed in the recently solved crystal structure of human MAdCAM-I give insight into the integrin binding preference of this unique receptor. Ki,iiiorrl\ MAdCAM-1, VCAM-I. ICAM-I. IgCAM, mAb, MEM. FCS Ahhwiitr/iolr.\. MAdCAM-I. Mucosnl addressin cell adhesion molecule-I: VCAM-I. Vasculnr cell adhesion molecule-I: ICAM-I. IgCAM. Imniunoglobulin like cell adhehion molecules: niAb. monoclonal antibody: MEM. minimal essential media; FCS, fetal calf SCI uill