The surface proteins of cultured human skin fibroblasts were iodinated and then exposed to one or more of the following blood coagulation proteins: thrombin, fibrinogen, and factor XIII (plasma protransglutaminase). Radiolabeled polypeptides were analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. After exposure to physiological concentrations of activated factor XIII (XIIIa), the band of radioactivity corresponding to the major labeled surface protein (fibronectin, molecular weight = 2.2 x 10(5) daltons) was cross-linked to a very high molecular weight complex. The cross-linking reaction was inhibited by fibrin (which is known to bind the catalytic subunit of XIIIa). Cross-linking of labeled cell surface fibronectin to fibrin could not be demonstrated. The fibrillar pattern of surface fibronectin appeared unaffected by cross-linking when studied by immunofluorescence. Cross-linking of cell surface fibronectin by XIIIa requires highly specific enzyme-substrate and protein-protein interactions, and may be an important physiological reaction.