LFA-1 and Mac-1, two β2integrin members constitutively expressed on neutrophils, mediate leukocyte recruitment cascade by binding to the same ligand of ICAM-1. The slow rolling and firm adhesion of leukocytes rely on LFA-1 while the cell crawling is dependent on Mac-1. We hypothesized that their distinct roles are likely attributed to the differences in the binding kinetics or in the diverse responses of outside-in and inside-out signaling. In this study, we compared the ICAM-1 binding features between soluble or membrane-expressed LFA-1 and Mac-1 with different affinity conformation using optical trap technique. Our data indicated that the affinity up-regulation from wide type(WT) to high affinity(HA) is off-rate dependent for LFA-1but on-rate dependent for Mac-1. The structural bases of this new finding were found to be consistent with our previous simulations. These results furthered our understanding in their function differences under shear flow.