Fibronectin is a large multidomain protein of the extracellular matrix that harbors two heparin binding sites, Hep-I and Hep-II, which support the heparin-dependent adhesion of melanoma and neuroblastoma cells [Barkalow, F. J. B., and Schwarzbauer, J. E. (1991) J. Biol. Chem. 266, 7812-7818; McCarthy, J. B., et al. (1988) Biochemistry 27, 1380-1388; Drake, S. L., et al. (1993) J. Biol. Chem. 268, 15859-15867]. The stronger heparin/HS binding site on fibronectin, Hep-II, spans fibronectin type III domains 12-14. Previous site-directed mutagenesis, nuclear magnetic resonance (NMR) chemical shift perturbation, and crystallographic structural studies all agree that the main heparin binding site is located on the surface of fibronectin type III domain 13 [Ingham, K. C., et al. (1993) Biochemistry 32, 12548-12553; Sharma, A., et al. (1999) EMBO J. 18, 1468-1479; Sachchidanand, L. O., et al. (2002) J. Biol. Chem. 277, 50629-50635]. However, the "synergy site" for heparin binding located on fibronectin type III domain 14 remained elusive because the actual binding sites could not be identified. Using NMR spectroscopy and isothermal titration calorimetry, we show here that heparin is able to bind to a cationic 'cradle' of fibronectin type III domain 14 formed by the PRARI sequence, which is involved in the integrin α4β1 interaction [Mould, A. P., and Humphries, M. J. (1991) EMBO J. 10, 4089-4095], and to the flexible loop comprising residues KNNQKSE between the last two β-strands, D and E, of FN14. Our data reveal that the individual FN14 domain binds to the sulfated sugars Dp8 and Reviparin with affinities similar to those of the individual domain FN13 [Breddin, H. K. (2002) Expert Opin. Pharmacother. 3, 173-182]. It is noteworthy that by introduction of the last β-strand of FN13 and the linker region between FN type III domains 13 and 14, the perturbation of NMR chemical shifts by heparin is significantly reduced, especially at the PRARI site. This indicates that the Hep-II binding site of fibronectin is mainly located on FN13 and the synergistic binding site on FN14 involves only the KNNQKSE sequence.