The Ig superfamily (IgSF) intercellular adhesion molecule-1 (ICAM-1) equilibrates between monomeric and dimeric forms on the cell surface, and dimerization enhances cell adhesion. A crystal structure of ICAM-1 IgSF domains (D) 3-5 revealed a unique dimerization interface in which D4s of two protomers fuse through edge -strands to form a single super -sandwich domain. Here, we describe a crystal structure at 2.7-Å resolution of monomeric ICAM-1 D3-D5, stabilized by the monomer-specific Fab CA7. CA7 binds to D5 in a region that is buried in the dimeric interface and is distal from the dimerization site in D4. In monomeric ICAM-1 D3-D5, a 16-residue loop in D4 that is disordered in the dimeric structure could clearly be traced as a BC loop, a short C strand, and a CE meander with a cis-Pro followed by a solvent-exposed, flexible four-residue region. Deletions of 6 or 10 residues showed that the C-strand is essential for monomer stability, whereas a distinct six-residue deletion showed little contribution of the CE meander. Mutation of two inward-pointing Leu residues in edge -strand E to Lys increased monomer stability, confirming the hypothesis that inward-pointing charged side chains on edge -strands are an important design feature to prevent -supersheet formation. Overall, the studies reveal that monomer-dimer transition is associated with a surprisingly large, physiologically relevant, IgSF domain rearrangement.crystal structure ͉ leukocytes ͉ flow cytometry ͉ mutagenesis I ntercellular adhesion molecule-1 (ICAM-1; CD54) is perhaps the most important member of a family of related Ig superfamily (IgSF) molecules that serve as ligands for the integrins ␣ L  2 , ␣ M  2 , and ␣ X  2 (1). ICAM-1 is expressed on the surface of cells important in immune responses. Inflammatory mediators further enhance expression of ICAM-1 on these cells and induce it on other cell types, including endothelial, epithelial, and fibroblastic cells. Increased ICAM-1 expression augments immune responses and leukocyte accumulation in inflamed tissues.ICAM-1 consists of five extracellular IgSF domains (D1-D5), a hydrophobic transmembrane domain, and a short cytoplasmic domain (1). In its native state on the cell surface ICAM-1 is in equilibrium between a monomeric and dimeric state (2, 3). Chemical cross-linking reveals a substantial proportion of dimeric material (2, 3). The fraction of monomeric cell-surface ICAM-1 can be estimated by using a mAb termed CA7 that binds to D5 of ICAM-1 (4) and binds much better to monomeric than dimeric ICAM-1 (2). The transmembrane domain of ICAM-1 stabilizes dimerization. ICAM-1 with the transmembrane domain replaced with a glycosylphosphatidylinositol (GPI) anchor is largely monomeric, whereas wild-type ICAM-1 is largely dimeric (2, 3). Recombinant soluble ICAM-1, lacking the transmembrane and cytoplasmic domains, exists as a monomer in solution (2, 4, 5).Crystal structures of D1-D2 and D3-D5 fragments of ICAM-1 have revealed two sites for dimerization, in D1 and D4. A dimerization site on the BE...