The lone 1F2 2F2 modular pair of fibronectin is found in the collagen- binding region. This exclusive localization suggests the 1F2 2F2 pair plays an important role in the recognition of collagen. However, no information is currently available about the interaction between the two F2 modules and, thus, the orientation of their putative collagen-binding sites with respect to one another. Comparison of a variety of high-resolution NMR parameters from the F2 modules in isolation and the 1F2 2F2 pair was used to establish the extent of interaction between the F2 modules in the pair. Chemical shifts of the F2 modules and the 1F2 2F2 pair indicate that the structures of the modules are preserved in the pair and that, with the exception of the covalent linkage, they do not interact. 15N NMR relaxation data identify significant motion occurring in the linker region of the 1F2 2F2 pair, and analyses of the anisotropic diffusion properties of the 1F2 2F2 pair are consistent with the modules in the F2 pair tumbling independent of one another.