Cryo-high-resolution scanning electron microscopy was used to analyze the conformation of fibronectin fibrils formed in human skin fibroblast cultures or in a cell-free system by treating soluble plasma fibronectin with guanidine. Structurally, fibrils assembled in the cell-free system and in culture were similar. Assembly of both fibrillar networks involves interactions with the III1 and amino terminal repeats of fibronectin; their conformations consist of either smooth surfaces or patches of smooth surfaces and nodules randomly spaced along the fibril. The random distribution of these two conformations in fibrils indicates that fibronectin fibrils are capable of undergoing localized conformational changes. The nodules may be discrete domains of 3 to 4 type III repeats, as they can be labeled with the monoclonal antibody IST-2 to the III13-14 repeats in fibronectin and are found in 160 kDa and 85 kDa fragments of fibronectin that only contain type III repeats. In our study, smooth regions of fibrils were never recognized by the IST-2 antibody, suggesting that the epitope in the III13-14 repeats is masked in these regions. These results demonstrate that fibronectin fibrils are flexible and certain epitopes of fibronectin may be buried, or exposed, depending on the conformation of the fibril. They also show that fibrils assembled in cell-free conditions can be a powerful tool for studying fibril formation.