Activated lymphocyte function-associated antigen-1 (LFA-1, αLβ2 integrin) found on leukocytes facilitates firm adhesion to endothelial cells by binding to intercellular adhesion molecule-1 (ICAM-1), which is upregulated on the endothelium at sites of inflammation. Recent work shows that LFA-1 in a preactivation, low-affinity state may also be involved in the initial tethering and rolling phase of the adhesion cascade. The ligand binding epitope of LFA-1 is contained in the inserted (I) domain, and a conformational change in this region during activation increases ligand affinity. We have displayed wild-type I domain on the surface of yeast and validated expression using I domain specific antibodies and flow cytometry. Surface display of I domain supports yeast rolling on ICAM-1-coated surfaces under shear flow. I domain mutants were also expressed, and soluble ICAM-1 binding studies validated that these mutants have a range of affinities for the ligand. Expression of the locked open, high-affinity I domain mutant supports firm adhesion of yeast under shear flow, while yeast displaying intermediate-affinity I domain mutants exhibit a range of rolling phenotypes. Rolling behavior for these mutants fails to correlate with ligand binding affinity. These results indicate that unstressed binding affinity is not the only molecular property that determines adhesive behavior under shear flow. Work to validate this observation on a larger scale is underway utilizing I domain mutant libraries to characterize the molecular structure-function relationship via directed evolution. Understanding of adhesive behavior of particles on ICAM-1 surfaces will facilitate further engineering of LFA-1 I domain for inflammatory targeting applications.