Surface coating of synthetic materials is often considered to improve biomedical devices biocompatibility. In this study, we covalently bound fibronectin (FN) onto ammonia plasma-treated PTFE via two crosslinkers, namely glutaric anhydride (GA) and sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-SMPB). With respect to clean PTFE, cell adhesion increased markedly on both FN grafted surfaces, although it was twice higher on PTFE-GA-FN than on PTFE-SMPB-FN. ELISA experiments performed with a polyclonal antibody revealed that the amount of FN is identical on both surfaces while monoclonal antibody specific to the RGD binding site clearly demonstrated a greater availability when FN is surface grafted through GA. These results provide evidence of a variation in protein conformation correlated with the surface conjugation strategy.