alpha(4) integrins play a pivotal role in leukocyte migration and tissue-specific homing. The ability of integrins to bind ligand is dynamically regulated by activation-dependent conformational changes triggered in the cytoplasmic domain. An NMR solution structure defined a putative membrane-proximal salt bridge between the alpha(IIb)beta(3) integrin cytoplasmic tails, which restrains integrins in their low-affinity state. However, the physiological importance of this salt bridge in alpha(4) integrin regulation remains to be elucidated. To address this question, we disrupted the salt bridge in murine germ line by mutating the conserved cytoplasmic arginine R(GFFKR) in alpha(4) integrins. In lymphocytes from knock-in mice (alpha(4)-R/A(GFFKR)), alpha(4)beta(1) and alpha(4)beta(7) integrins exhibited constitutively up-regulated ligand binding. However, transmigration of these cells across VCAM-1 and MAdCAM-1 substrates, or across endothelial monolayers, was reduced. Perturbed detachment of the tail appeared to cause the reduced cell migration of alpha(4)-R/A(GFFKR) lymphocytes. In vivo, alpha(4)-R/A(GFFKR) cells exhibited increased firm adhesion to Peyer patch venules but reduced homing to the gut. Our results demonstrate that the membrane-proximal salt bridge plays a critical role in supporting proper alpha(4) integrin adhesive dynamics. Loss of this interaction destabilizes the nonadhesive conformation, and thereby perturbs the properly balanced cycles of adhesion and deadhesion required for efficient cell migration.