Alternative splicing of the fibronectin (FN) gene transcript provides an efficient mechanism for generating functionally appropriate forms of this adhesive glycoprotein, in situ. Cellular FNs that include the EIIIA and/or EIIIB FN III segments are prominently expressed during embryogenesis, wound healing, tumor progression and inflammation. However, the roles of this domain in altering overall FN protein structure and regulating cellular function remain unclear. We previously reported that two integrins, 91 and 41, ligate the EIIIA segment (1) and that the epitopes for function-blocking monoclonal antibodies lie within the C-C’ loop of EIIIA (2). We have now performed site-directed mutagenesis within the EIIIA segment and carried out cell adhesion assays on these mutant EIIIAs. We find that the Asp41 and Gly42 residues within the C-C’ loop of EIIIA are necessary for integrin 91 binding. Synthetic peptides based on the predicted important amino acid sequence from the C-C’ loop encode sufficient information to completely inhibit 91-mediated cell adhesion. We also report that EIIIA promotes filopodial formation in 91-expressing cells accompanied by Cdc42 activation. Our data provide a cellular activity for the EIIIA segment, evidence for conformational lability and peptide sequences for probing EIIIA functions in vitro and in vivo.