Assembly of a fibronectin (FN) matrix is a multistep process which influences a number of cellular functions including intracellular cytoskeletal organization and signaling responses. We have previously reported on a recombinant FN (recFN), FN delta III1-7, which differs from native FN in its rate of fibril formation. To determine the intracellular consequences of a delay in assembly, we compared the distribution of cytoskeletal proteins during the formation of native and recFN matrices by immunofluorescence at various time points. CHO alpha 5 cell cytoskeleton was reorganized in response to both native and recFN matrix formation. Assembly of native FN induced a rapid reorganization of actin into stress fibers and colocalization of alpha 5 beta 1 integrin, focal adhesion kinase (FAK), vinculin, and paxillin to regions of cell-matrix contact. alpha 5 beta 1 integrins and FAK are also clustered upon binding of FN delta III1-7 to cells but actin reorganization and focal adhesion formation are delayed and appear to be dependent on the formation of FN delta III1-7 fibrils. These results suggest that the structural framework of the matrix plays an important role in the ability of FN to initiate intracellular responses.