The major antigen derived from elastic fiber microfibrils was identified as a Mr = 31,000 glycoprotein, using immunoblotting and immunohistochemical techniques with antisera raised to "reductive guanidine extracts" of fetal bovine nuchal ligament, and to subfractions of these. A second, elastic fiber-derived, but unidentified, antigen of large molecular size (Mr greater than 200,000) was present in these extracts. Antisera raised to the purified 31-kDa glycoprotein were shown, by immunoelectron microscopy, to localize specifically to the elastin-associated microfibrils. Thus, the macro-molecule was called "microfibril-associated glycoprotein" or MAGP. MAGP is an acidic glycoprotein with a distinctive amino acid composition, being exceptionally rich in glutamic acid, rich in cystine, and low in glycine. MAGP was extractable from tissue homogenates using NaCl, urea, or guanidine hydrochloride solutions, only if a strong reducing agent was present. Thus, disulfide bonding is important for the strong association of MAGP with elastic fibers. Immunoblotting with anti-MAGP antiserum identified two additional reactive species, of Mr = 60,000 and Mr approximately 300,000, in tissue extracts. As only the 31-kDa species was detected in fibroblast culture medium, these additional species were probably aggregates, rather than precursors. MAGP did not react with antilysyl oxidase antiserum on immunoblots or by enzyme-linked immunosorbent assay. MAGP is the first macromolecule to have been established to be a constituent of elastin-associated microfibrils in both developing and mature elastic tissues.