Ephrins are cell surface ligands that activate Eph receptor tyrosine kinases. This ligand-receptor interaction plays a central role in the sorting of cells. We have previously shown that the ephrinB-EphB signalling pathway is also involved in the migration of intestinal precursor cells along the crypts. Using the colon cell line DLD1 expressing the EphB2 receptor, we showed that stimulation of these cells with soluble ephrinB1 results in a rapid retraction of cell extensions and a detachment of cells. On ephrinB1 stimulation, the small GTPases Rho and Ras are activated and Rap1 is inactivated. Importantly, when a constitutively active Rap1 mutant was introduced into these cells, ephrinB1-induced retraction was inhibited. From these results, we conclude that down-regulation of Rap1 is a prerequisite for ephrin-induced cell retraction in colon cells.