Cephalic neural crest (NC) cells enter a cell-free space (CFS) that contains an abundant extracellular matrix (ECM). Numerous in vitro investigations have shown that extracellular matrices can influence cellular activities including NC cell migration. However, little is known about the actual ECM composition of the CFS in vivo, how the components are distributed, or the nature of NC cell interactions with the CFS matrix. Using ultrastructural, autoradiographic, and histochemical techniques we analyzed the composition and spatial organization of the ECM found in the CFS and its interaction with mesencephalic NC cells. We have found that a specific distribution of glycoproteins and sulfated polyanions existed within the CFS prior to the translocation of NC cells and that this ECM was modified in areas occupied by NC. The interaction between the ECM components and the NC cells was not the same for all NC cells in the population. Subpopulations of the NC cell sheet became associated with ECM of the ectoderm (basal lamina) while other NC cells became associated with the ECM of the CFS. Trailing NC cells (NC cells that emerge after the initial appearance of NC cells) encountered a modified ECM due to extensive matrix modifications by the passage of the initial NC cell population.