Current models of the elastic properties and structural organization of fibrillin-containing microfibrils are based primarily on microscopic analyses of microfibrils liberated from connective tissues after digestion with crude collagenase. Results presented here demonstrate that this digestion resulted in the cleavage of fibrillin-1 and loss of specific immunoreactive epitopes. The proline-rich region and regions near the second 8-cysteine domain in fibrillin-1 were easily cleaved by crude collagenase. Other sites that may also be cleaved during microfibril digestion and extraction were identified. In contrast to collagenase-digested microfibrils, guanidine-extracted microfibrils contained all fibrillin-1 epitopes recognized by available antibodies. The ultrastructure of guanidine-extracted microfibrils differed markedly from that of collagenase-digested microfibrils. Fibrillin-1 filaments splayed out, extending beyond the width of the periodic globular beads. Both guanidine-extracted and collagenase-digested microfibrils were subjected to extensive digestion by crude collagenase. Collagenase digestion of guanidine-extracted microfibrils removed the outer filaments, revealing a core structure. In contrast to microfibrils extracted from tissues, cell culture microfibrils could be digested into short units containing just a few beads. These data suggest that additional cross-links stabilize the long beaded microfibrils in tissues. Based on the microfibril morphologies observed after these experiments, on the crude collagenase cleavage sites identified in fibrillin-1, and on known antibody binding sites in fibrillin-1, a model is proposed in which fibrillin-1 molecules are staggered in microfibrils. This model further suggests that the N-terminal half of fibrillin-1 is asymmetrically exposed in the outer filaments, whereas the C-terminal half of fibrillin-1 is present in the interior of the microfibril.Microfibrils have been extracted from a variety of tissues and visualized by electron microscopy as distinctive beaded string structures (1, 2). The molecular composition of these structures is assumed to be complex. However, based upon immunolocalization of fibrillin to these structures (2, 3) and the shape of fibrillin monomers (4, 5), fibrillins are thought to be the major backbone components of these extracted microfibrils. Fibrillincontaining microfibrils are ubiquitous in the connective tissue space (6), providing architectural support as well as information essential for appropriate signaling during morphogenesis (7-9). In human disorders associated with fibrillins, the structural integrity of fibrillin microfibrils is required for the proper function of certain connective tissues (10). Therefore, determination of the organization of fibrillin molecules within the microfibril is important basic information.The organization of fibrillin molecules within microfibrils has been controversial. Both parallel (head-to-tail) (3, 4) and antiparallel (11) arrangements of fibrillin molecules within microfibrils have been...