Cellular adhesion to fibronectin (FN) can be mediated by several sequences located in different portions of the molecule. In human FN, these are: (i) the bipartite RGDS domain containing the RGDS cell-binding sequence functioning in synergy for full cellular adhesion with a second site (termed here the synergistic adhesion site) and (ii) the recently characterized CS1 and REDV adhesion sites within the alternatively-spliced type III homology-connecting segment. Using specific adhesive ligands and inhibitory probes, we have examined the role of each of these domains in the adhesion, spreading, and motility of avian neural crest cells in vitro. Both the RGDS domain and the CS1 adhesion site were found to promote attachment of neural crest cells, but only the RGDS domain supported their spreading. However, the RGDS sequence could mediate both attachment and spreading efficiently only when it was associated with the synergistic adhesion site. In migratory assays, it was found that both the RGDS domain and the CS1 site are required in association, each with functional specificity, to permit effective locomotion of neural crest cells. The REDV adhesion site was apparently not recognized by avian neural crest cells, presumably because this sequence is absent from chicken FN. Finally, it was found that recognition of both the RGDS domain and CS1 binding site by neural crest cells involved receptors belonging to the integrin family. From these results, we conclude that neural crest cells can interact with several binding sites of FN molecules, and use them for distinct functions. Our results also suggest the possibility of an instructive role for FN in the control of adhesive and migratory events during embryonic development.