The interaction of integrin alpha(4)beta(1) with endothelial VCAM-1 controls the trafficking of lymphocytes from blood into peripheral tissues. Cells actively regulate the affinity of alpha(4)beta(1) for VCAM-1 (activation). To investigate the biological function of alpha(4)beta(1) activation, we isolated Jurkat T cell lines with defective alpha(4)beta(1) activation. Using these cells, we found that alpha(4)beta(1)-stimulated alpha(L)beta(2)-dependent cell migration was dramatically reduced in cells with defects in alpha(4)beta(1) activation. These cells required 20 times more VCAM-1 to promote alpha(L)beta(2)-dependent cell migration. This defect was at the level of alpha(4)beta(1) affinity as an activating alpha(4)beta(1) Ab rescued alpha(4)beta(1)-stimulated alpha(L)beta(2)-dependent migration. In contrast, migration of alpha(4)beta(1) activation-defective cells on VCAM-1 alone was enhanced at higher VCAM-1 densities. Thus, alpha(4)beta(1) activation determines a set point or threshold at which VCAM-1 can regulate alpha(L)beta(2)-dependent as well as alpha(4)beta(1)-dependent cell migration. Changes in this set point may specify preferred anatomical sites of integrin-dependent leukocyte emigration from the bloodstream.