Soluble fibronectin is found in body fluids and media of adherent cultured cells and binds to fibrin and collagen. Insoluble fibronectin is found in tissue stroma and in extracellular matrices of cultured cells. Fibronectin is a substrate for Factor XIIIa (plasma transglutaminase) and can be cross-linked by Factor XIIIa to itself and the the alpha-chain of fibrin. We used sodium dodecyl sulfate-polyacrylamide gel electrophoresis to investigate Factor XIIIa-mediated crosslinking of fibronectin to collagen. At O degrees or 37 degrees C, fibronectin could be cross-linked to iodinated cyanogen bromide fragment 7 of the alpha 1(I) chain. At 22 degrees or 37 degrees C, fibronectin could be cross-linked to isolated alpha 1(I) chains of type I collagen. Fibronectin could also be crosslinked to types I and III collagen, but only at 37 degrees C. alpha 1(I)-CB7, alpha 1(I) collagen chains, type I collagen, type III collagen, and fibrin all blocked cross-linking between 125I-alpha 1 (I)-CB7 and fibronectin. alpha 1(I)-CB7 blocked cross-linking between fibronectin and fibrin. These results indicate that the determinants of fibronectin-fibrin and fibronectin-collagen binding and cross-linking are similar. Cross-linking of fibronectin to collagen likely occurs in vivo and may be important for normal wound healing, collagen fibrillogenesis, and embryogenesis.