Interactions between type III domains within the cell-binding region of fibronectin have been deduced through a study of the thermal stability of the 110 kDa cell-binding fragment and a variety of its subfragments by scanning calorimetry and fluorescence spectroscopy. Comparison of the melting profiles of different fragments demonstrated that all type III modules comprise independently folded domains and revealed that in the parent 110 kDa fragment, domains 2, 4, 5, 8, 9 and 11 are relatively labile (tm near 60 degrees C) while 3, 6, 7 and 10 are thermostable (tm above 110 degrees C). Three types of interactions were found: (1) stabilizing interactions, manifested by a decrease in the tm of one of the interacting domains when they are separated; (2) destabilizing interactions for which tm is elevated upon separation; and (3) cooperative interactions in which two adjacent domains tend to melt together in a single two-state transition. Examples of the latter include the pairs 4-5 and 8-9. Long range stabilizing interactions occurred primarily between thermostable domains, 3 with 6, 6 with 7 and 10, and 7 with 9. For example, module 9, which contains the synergistic cell-binding site, is destabilized by 16 degrees C in the absence of module 7. These long range interactions, some of which could be disrupted by alternative splicing, undoubtedly influence the supertertiary structure of the central region of fibronectin, rendering it more compact than previously appreciated and possibly playing a role in the regulation of matrix assembly or interactions with other molecules such as integrin receptors on cell surfaces.