Assembly of fibronectin fibrils occurs at the surface of substrate-attached cells and is mediated by the first to the fifth type I modules in the N-terminal 70 kDa portion of the molecule. The first type III module (III₁) of fibronectin, not present in the 70 kDa portion, contains a conformation-dependent binding site for the 70 kDa N-terminal region of fibronectin, suggesting that the III₁ module on cell-surface fibronectin may serve as a binding site for fibronectin's N-terminus on substrate-attached cells. To explore this possiblility, we compared the ability of mutant recombinant 70 kDa proteins containing deletions of one or several of the first five type I modules to bind to fibroblasts and to III₁. Proteins containing the fourth and fifth type I modules (70KΔI_1-3) bound specifically to III₁ in solid-phase binding assays; proteins lacking I₄ and I₅ did not bind. N-terminal molecules containing the fourth and fifth type I modules also bound to fibroblasts, suggesting that III₁-like binding sites are present on the cell surface. However, the high-affinity binding sites on fibroblasts for fibronectin or the 70 kDa protein displayed more complex determinants, inasmuch as 70 kDa deletion mutants lacking I₄ and I₅ also bound to the cell surface, and deletion mutants lacking I_1-3 and I_4-5 both competed only partially for binding of ¹²⁵I-labelled fibronectin or 70 kDa protein. These data indicate that the N-terminal part of fibronectin binds to III₁ via I₄ and I₅ and that interactions in addition to that of I₄ and I₅ with III₁ are important for cell-surface-mediated fibronectin polymerization.