The restriction of intermingling between specific cell populations is crucial for the maintenance of organized patterns during development. A striking example is the restriction of cell mixing between segments in the insect epidermis and the vertebrate hindbrain that may enable each segment to maintain a distinct identity. In the hindbrain, this is a result of different adhesive properties of odd- and even-numbered segments (rhombomeres), but an adhesion molecule with alternating segmental expression has not been found. However, blocking experiments suggest that Eph-receptor tyrosine kinases may be required for the segmental restriction of cells. Eph receptors and their membrane-bound ligands, ephrins, are expressed in complementary rhombomeres and, by analogy with their roles in axon pathfinding, could mediate cell repulsion at boundaries. Remarkably, transmembrane ephrins can themselves transduce signals, raising the possibility that bi-directional signalling occurs between adjacent ephrin- and Eph-receptor-expressing cells. We report here that mosaic activation of Eph receptors leads to sorting of cells to boundaries in odd-numbered rhombomeres, whereas mosaic activation of ephrins results in sorting to boundaries in even-numbered rhombomeres. These data implicate Eph receptors and ephrins in the segmental restriction of cell intermingling.