Small intestinal submucosa (SIS) is an acellular biomaterial derived from porcine jejunum. When used as a soft tissue graft material, SIS induces site-specific remodeling of the organ or tissue in which it is placed. The mechanism by which SIS induces tissue remodeling is only partially understood. Fibronectin (Fn) is a dimeric glycoprotein present in plasma and extracellular matrix. Fn exhibits chemotactic and adhesive properties for many cells, including fibroblasts and endothelial cells. Thus, Fn may play a key role in the tissue-remodeling activity of SIS. The goals of this study were to localize and quantify the Fn in SIS, and characterize the structure of SIS-derived Fn. Immunohistochemical staining confirmed the transmural presence of Fn in SIS. The Fn content of SIS was 0.08 ± 0.05 % dry weight, similar to the Fn content of other similar tissues, as determined with a competitive ELISA. SDS-PAGE and Western blots of purified protein showed SIS- derived Fn to migrate similar to human and porcine plasma fibronectins. Fn derived from SIS did not contain domains associated with embryonic or transitional extracellular matrix. Fn is transmurally distributed throughout the thickness of SIS and its content and structure are consistent with stable, mature tissue.