Microfibrils were dissected from the zonular apparatus of the bovine eye, homogenized and used as an immunogen to prepare monoclonal antibodies. Initial screening of hybridomas was performed by immunoblotting to a sonicate of zonular fibrils and by immunolocalization to frozen sections of the zonular apparatus. Subsequently, monoclonal antibodies with strong immunoreactivity to zonular fibrils were shown to recognize microfibrils in a wide range of connective tissues both by immunofluorescent staining and by electron microscopic immunolocalization. All antibodies were found to recognize a single protein of 350 kDa on Western blotting of the proteins secreted by bovine aortic smooth muscle cells. A protein of the same molecular weight and properties was recognized by an antibody previously prepared by another group against fibrillin. A member of the fibrillin family therefore represents the major immunogen of intact zonular fibrils, and the results support previous evidence for a close relationship between zonular fibrils and other connective tissue microfibrils. The zonular apparatus is a suitable system to obtain purified preparations of microfibrils in order to investigate their composition and structural organization.