Fibronectin adsorbs to a variety of surfaces. Adsorption on different biomaterial substrates can result in different conformations for the adsorbed fibronectin. The adsorption isotherms of fibronectin to either neutral polystyrene or negatively charged polystyrene are indistinguishable. Sulfonation was used to vary the level of negative charge on polystyrene surfaces, and adhesion strength of HT1080 cells to these surfaces following fibronectin coating was measured using a spinning disk device. Adhesion to the different fibronectin surfaces was alpha5beta1 integrin dependent, and charge variation had little effect on overall adhesion strength. Ligation of alpha5beta1 integrin to surface-bound fibronectin resulted in a fibronectin-density-dependent increase in phosphorylation of FAK Y397 when negatively charged substrates were used but resulted in no FAK phosphorylation when uncharged substrates were used. Similarly, cell spreading was dependent on the level of negative charge. Thus, the use of specific biomaterial substrates can separate the adhesion and signaling functions of alpha5beta1 integrin. Polylysine coating was used to analyze the effect of positive charge. On the polylysine substrate, there is an increase in the level of nonspecific adhesion following coating with fibronectin and/or bovine serum albumin (BSA). On BSA substrates, there was no stimulation of FAKY397 phosphorylation. Addition of fibronectin to the polylysine surfaces produced a weak stimulation of FAK Y397 phosphorylation indicating that the positively charged surface was also unfavorable for supporting alpha5beta1-mediated signals.