Hindbrain segments, rhombomeres, define distinct cellular and molecular domains which furnish the ground plan for important aspects of neural and cranial development. In this study, further evidence is presented that the interfaces between rhombomeres, rhombomere boundaries, contain both cells and extracellular matrix with specialised characteristics. Cells at rhombomere boundaries show temporally and spatially distinct expression patterns of developmentally important genes. Towards the end of the developmental period when rhombomeres are present, a fan-shaped array of cells at rhombomere boundaries, that constitute the ventricular ridge, shows decreased expression of two genes (Hoxb-1 and Krox-20), which earlier in development were expressed in all cells of specific rhombomeres. In contrast, these boundary cells show increased expression of another gene, Pax-6, which earlier in development has a rhombomere-specific expression pattern. A specialised identity for boundary cells is further suggested by increased labelling with an anti-vimentin antibody at rhombomere boundaries, indicating that at least some boundary cells are radial glia or glial precursors. In addition to distinct cellular properties, the extracellular domain at rhombomere boundaries is also specialised. Chondroitin sulphate proteoglycan (CSPG) immunoreactivity is increased and, as revealed by immuno-electron microscopy, localised to extracellular spaces. CSPG is also enriched in boundaries regenerated after ablation, or boundaries generated ectopically by rhombomere transplantation. We propose that rhombomere boundaries form their characteristic morphology at the interface between groups of cells with differing molecular characteristics, representing different cell states. A specialised band of cells then develops at the interface. Both the boundary cells and extracellur matrix have characteristics which could be important in later events of neural development such as axon guidance and cell migration.