Fibronectin matrix assembly is thought to involve binding interactions between the amino-terminal I1-5 repeats and the first type III repeat (III1). Here we report that a third site, located within the III12-14 repeats of the carboxyl-terminal heparin II domain of fibronectin, is also involved in fibrillogenesis. Heparin II fragments inhibited fibril formation and binding of 125I-labeled fibronectin and/or 70-kDa fragments to the cell surface, deoxycholate-insoluble matrix, and adsorbed 160-kDa cell adhesion fragments of fibronectin. The inhibitory effects of heparin II fragments were as large or up to 20 times larger than those of a 44-kDa fibronectin fragment containing the III1 repeat. Under physiological conditions, amino-terminal fragments of fibronectin containing the I1-5 repeats interacted preferentially with proteolytically derived heparin II fragments and a recombinant III12-14 peptide both in solution and in solid phase, indicating that matrix assembly may involve direct interactions between I1-5 and III12-14 repeats. Interactions between the I1-5 repeats and 160-kDa fragments containing the III12-14 and III1 repeats could be inhibited by >/= 90% by either an anti-III13-14 monoclonal antibody (mAb) (IST-2) or an anti-III1 mAb (9D2), suggesting that cooperative interactions between III12-14 and III1 repeats may also promote binding of the I1-5 repeats. Neither mAb IST-2 nor mAb 9D2, alone or in combination, inhibited binding of 125I-labeled 70-kDa fragments to cycloheximide-treated cells plated on the 160-kDa substrate, suggesting that additional I1-5 binding sites, independent of the III1 and III12-14 repeats, may be involved in fibrillogenesis.