Protein adsorption onto synthetic materials influences cell adhesion and signaling events that direct cell function in numerous biomedical applications. Adsorption of fibronectin (FN) to different surfaces alters protein structure and modulates alpha(5)beta(1) integrin binding, cell adhesion, cell spreading, and cell migration. In the present study, self-assembled monolayers of alkanethiols on Au were used to analyze the effects of surface chemistry (CH3, OH, NH2, and COOH) on the adsorption of a recombinant fragment of FN, FNIII7-10, that incorporates both the synergy and RGD cell binding motifs. Surface chemistry potentiated differential FNIII7-10 adsorption kinetics and adsorbed structure as determined by surface plasmon resonance spectroscopy and antibody binding assays. FNIII7-10 functional activity, determined by cell adhesion strength, was modulated in a fashion consistent with these structural changes (OH = NH2 > COOH > CH3). However, these changes in protein parameters did not correlate simply to differences in surface hydrophobicity, indicating that additional surface parameters influence protein adsorption. These results demonstrate that surface chemistry modulates adsorbed protein structure and activity and establish a relationship between surface-dependent changes in structural domains of FNIII7-10 and functional activity.