Initiation of fibronectin (FN) matrix assembly is dependent on specific interactions between FN and cell surface integrin receptors. Here, we show that de novo FN matrix assembly exhibits a slow phase during initiation of fibrillogenesis followed by a more rapid growth phase. Mn2+, which acts by enhancing integrin function, increased the rate of FN fibril growth, but only after the initial lag phase. The RGD cell-binding sequence in type III repeat 10 is an absolute requirement for initiation by alpha5beta1 integrin. To investigate the role of the cell-binding synergy site in the adjacent repeat III9, a full-length recombinant FN containing a synergy mutation, FN(syn-), was tested for its ability to form fibrils. Mutation of this site drastically reduced FN assembly by CHOalpha5 cells. Only sparse short fibrils were formed even after prolonged incubation, indicating that FN(syn-) is defective in progression of the assembly process. These results show that the synergy site is essential for alpha5beta1-mediated accumulation of a FN matrix. However, the incorporation of FN(syn-) into fibrils and the deoxycholate-insoluble matrix could be stimulated by Mn2+. Therefore, exogenous activation of integrin receptors can overcome the requirement for FN's synergy site as well as modulate the rate of FN matrix formation.