Many pathogenic Gram-positive bacteria express cell surface proteins that bind to components of the extracellular matrix. This paper describes studies of the interaction between ligand binding repeats (D3 and D1-D4) of a fibronectin-binding protein from Staphylococcus aureus with a module pair (4F15F1) from the N-terminal region of fibronectin. When D3 was added to isotope-labeled 4F15F1, 1H, 15N, and 13C NMR chemical shift changes indicate that binding is primarily via residues in 4F1, although a few residues in 5F1 are also affected. Both hydrophobic and electrostatic interactions appear to be involved. The NMR data indicate that part of the D3 repeat converts from a disordered to a more ordered, extended conformation on binding to 4F15F1. In further NMR experiments, selective reduction of the intensity of D1-D4 resonances was observed on binding to 4F15F1, consistent with previous suggestions that in each of D1, D2, and D3 repeats, the main fibronectin binding site is in the C-terminal region of the repeat. In D1-D4, these regions also appear to go from a disordered to a more ordered conformation of fibronectin binding. Although the regions of the two proteins which interact had been previously identified, the findings presented here identify, for the first time, the specific residues in both proteins that are likely to be involved in the interaction.