Shear promotes endothelial recruitment of leukocytes, cell activation, and transmigration. Mechanical stress on cells caused by shear can induce a rapid integrin conformational change and activation, followed by an increase in binding to the extracellular matrix. The molecular mechanism of increased avidity is unknown. We have shown previously that the affinity of the alpha(4)beta(1) integrin, very late antigen-4 (VLA-4), measured with an LDV-containing small molecule, varies with cellular avidity, measured from cell disaggregation rates. In this study, we measured in real time affinity changes of VLA-4 in response to shear. The resulting affinity was comparable with the state mediated by receptor signaling and corresponded in time with intracellular Ca(2+) responses. Ca(2+) ionophores and N,N'-[1,2-ethanediyl-bis(oxy-2,1-phenylene)]bis[N-[2-[(acetyloxy)methoxy]-2-oxoethyl]]-, bis[(acetyloxy)methyl]ester demonstrate that the affinity regulation of VLA-4 in the presence of shear was related to Ca(2+) signaling. Pertussis toxin treatment implicates G(i) in an unknown pathway that connects shear, Ca(2+) elevation, VLA-4 affinity, and cell avidity.