Fibronectin is an extracellular matrix molecule composed of repeating subunits that create functional domains. These domains contain multiple binding sites for heparin and for various cell-surface receptors that modulate cell function. To examine the role that the high affinity heparin-binding region and the alternatively spliced V region of fibronectin play in tumor invasion, we expressed and purified four complementary recombinant fibronectin proteins. These proteins either included or excluded the alternatively spliced V region and contained either a mutated, non-functional high affinity heparin-binding domain (Hep-) or an unmutated heparin-binding domain (Hep+). Cultured oral squamous cell carcinoma cells were assayed for invasion into a Matrigel/collagen matrix supplemented with these four purified recombinant proteins, and for spreading and motility on plastic. Increased invasion was observed in gels supplemented with the V-Hep+ protein when compared with the V-Hep- protein. Inclusion of the V region in the proteins enhanced the invasion and migration associated with both Hep+ and Hep- proteins, whereas cell spreading was enhanced with the Hep+ recombinant proteins. These data demonstrate that both the high affinity heparin-binding domain and the V region of fibronectin play important roles in invasion, motility, and spreading of oral squamous cell carcinoma cells.