The integrin receptor alpha 4 beta 1 (also known as VLA-4) binds two different ligands, the endothelial cell surface protein vascular cell adhesion molecule-1 (VCAM-1) and the extracellular matrix component fibronectin. Three distinct sites in fibronectin are recognized by alpha 4 beta 1. Two of these (represented by peptides CS1 and CS5) are present in the alternatively spliced IIICS region and lie in separate, independently spliced segments of this region. A third site resides in the adjacent constitutively expressed HepII domain. Recombinant proteins containing the HepII domain and different splice variants of the IIICS have been generated and compared for their ability to mediate cell attachment, spreading and migration. The activity of these proteins has also been compared with that of a recombinant soluble form of VCAM-1 (rsVCAM-1). All the recombinant proteins supported A375-SM human melanoma cell attachment and spreading in an alpha 4 beta 1-dependent manner, but had varied adhesive activities with rsVCAM-1 > fibronectin variants containing the CS1 sequence >> other fibronectin variants. Low concentrations of rsVCAM-1 and CS1-containing fibronectin variants effectively supported cell migration in a trans-filter assay; however, cell motility was retarded at high concentrations of the same proteins. Fibronectin variants lacking CS1 supported little or no migration. To obtain further insight into the molecular basis of this varied adhesive activity, apparent dissociation constants for each of the recombinant proteins were measured using a solid phase receptor-ligand binding assay. The results revealed a hierarchy of ligand affinities that mirrored their adhesive activity (rsVCAM-1 > fibronectin variants containing CS1 >> other fibronectin variants).