Fibronectin, a large modular protein, interacts with many other proteins in the extracellular matrix and on the cell surface. It has previously been shown that interactions between noncontiguous modules exist in the collagen binding region. It is shown here that the interaction between the sixth type I module (6F1) and the second type II module (2F2) can be disrupted by mutation of a residue in the intermodule interface of the 6F11F22F2 fragment. The perturbation of the interface and the binding of collagen-derived peptides to individual modules were assessed by high-resolution nuclear magnetic resonance (NMR) spectroscopy. Cooperativity between the modules in binding ligand was assessed by analytical gelatin affinity chromatography of the mutant and wild-type proteins. Differential scanning calorimetry (DSC) was used to probe the influence of the interface on module stability. It is shown that while the 6F1−2F2 interface confers significant thermal stability to the 2F2 module, it has little effect on gelatin binding activity of the 6F11F22F2 fragment.