The fibronectin matrix contains cryptic sites which are thought to modulate cellular biological responses. One of these sites, located in fibronectin's first type III repeat (III1c), influences signaling pathways that are relevant to cytoskeletal organization and cell cycle progression. The purpose of this study was to identify possible mechanisms responsible for the effects of III1c on cell behavior. Recombinant peptides representing various type III repeats of fibronectin were compared for their effects on fibronectin matrix organization and activation of intracellular signaling pathways. III1c and III13 but not III11c or III10 bound to monolayers of human skin fibroblasts in a dose- and time-dependent manner and were localized to the extracellular matrix. Binding of III13, but not III1c, to matrix was sensitive to heparitinase, suggesting that the association of III1c with the matrix was not dependent on heparan sulfate proteoglycans. Quantitative and morphological assessment indicated that, in contrast to previously published reports, the binding of III1c to cell layers did not result in the loss or disruption of matrix fibronectin. Binding of III1c but not III13 to the extracellular matrix did result in the loss of a conformationally sensitive epitope present within the EDA type III module of cellular fibronectin. III1c-induced loss of the EDA epitope did not require the presence of cells, occurred within 1 hour and was associated with the activation of p38 mitogen-activated protein kinase (MAPK) followed by the formation of filopodia. Maximal phosphorylation of p38 MAPK occurred within 1 hour, whereas cytoskeletal changes did not appear until 12 hours later. These findings are consistent with a model in which the binding of III1c to the extracellular matrix results in a conformational remodeling of the fibronectin matrix, which has both short- and long-term effects on cell physiology.