HT-29 colon carcinoma cells attach to TNFalpha-activated human umbilical vein endothelial cells (HUVECs) by their specific binding to E-selectin. This interaction activates, in the cancer cells, the MAPK SAPK2/p38, which leads to their transendothelial migration (Laferrière et al., J Biol Chem 2001; 276: 33762). In this study, we investigated the role of E-selectin in activating integrins to modulate adhesion and regulate integrin-mediated events. Blocking the integrins from HT-29 cells (alpha2, alpha3, alpha6, alphav/beta5, beta1 and beta4) with specific antibodies revealed a role for beta4 integrin in their adhesion to TNFalpha-treated HUVEC. The beta4 integrin-dependent adhesion was maximal after 30 min, whereas the-E-selectin-dependent adhesion was maximal after 15 min. Integrin beta4 became quickly phosphorylated upon addition of HT-29 cells to endothelial cells and the effect was independent of the expression of E-selectin. Moreover, a recombinant E-selectin/Fc chimera did not induce the phosphorylation of beta4. The phosphorylation of beta4 is not required for adhesion since adhesion was not affected in HT-29 cells that express a truncated form of beta4 that is deleted from its cytoplasmic phosphorylatable domain. However, the expression of the non-phosphorylatable deletant of beta4 was associated with decreased transendothelial cell migration underscoring the key role for the cytoplasmic domain of beta4 in cell migration. We suggest: 1) that the adhesion of HT-29 cells to activated endothelial cells follows at least two essential sequential steps involving the binding of E-selectin to its receptor on carcinoma cells and then the binding of beta4 to its own receptor on endothelial cells; 2) that the phosphorylation of integrin beta4 contributes to enhance the motile potential of cancer cells and increase their trans-endothelial migration. Overall, our results indicate that the interaction of metastatic cancer cells with endothelial cells implies a specific sequence of signaling events that ultimately leads to an increase in their efficient transendothelial migration.