Fibronectin (FN) matrix assembly is an integrin-mediated process that is regulated by both the extracellular environment and intracellular signaling pathways. The activity of Src-family kinases is important for initiation of FN assembly by normal fibroblasts. Here we report that in HT1080 fibrosarcoma cells, Src kinase activity is required not only for the assembly of FN matrix but also for the maintenance of FN matrix fibrils at the cell surface. Dexamethasone-induced FN fibril formation by these cells was completely blocked for at least 24 h when Src-family kinase activity was inhibited by either PP1 or SU6656. Inhibition of Src after significant matrix had already been assembled, resulted in an increased rate of loss of detergent-insoluble FN. Binding of activation-dependent integrin antibodies reveals a role for Src in maintaining integrin activity. The requirement for Src kinase activity appears to depend, in part, on phosphorylation of paxillin at tyrosine 118 (Y118). Phospho-paxillin co-localized with FN fibrils, and overexpression of GFP-paxillin but not of GFP-paxillinY118F enhanced cell-mediated assembly of FN. Our results indicate that Src maintains FN matrix at the cell surface through its effect on integrin activity and paxillin phosphorylation.