The study we present involves the use of a biomimetic system that allows us to study specific interactions in the alpha(5)beta(1) receptor-GRGDSP ligand system with an atomic force microscope (AFM). Bioartificial membranes that mimic the adhesion domain of the extracellular matrix protein fibronectin are constructed from peptide-amphiphiles. A novel peptide-amphiphile is designed that contains both GRGDSP (GlyArg-Gly-Asp-Ser-Pro, the primary recognition site for alpha(5)beta(1)) and PHSRN (Pro-His-Ser-Ar-Asn, the synergy binding site for alpha(5)beta(1))sequences in a single peptide formulation, separated by a spacer. Two different antibodies are used to immobilize and activate isolated a(5)beta(1) integrins on the AFM tip. The interaction measured between immobilized alpha(5)beta(1) integrins and peptide-amphiphiles is specific for integrin-peptide binding and is affected by divalent cations in a way that accurately mimics the adhesion function of the a(5)beta(1) receptor. The strength of the PHSRN synergistic effect depends on the accessibility of this sequence to alpha(5)beta(1) integrins. An increase in adhesion is observed compared to surfaces displaying only GRGDSP peptides when the new biomimetic peptide-amphiphiles are diluted with lipidated poly(ethylene glycol), which provides more space for the peptide headaroups to bend and expose more of the PHSRN at the interface.