Using Western immunoblotting, the extractable proteins of the bovine zonular fibers were examined for reactivity with two zonular anti-sera known to have strong affinity for zonular fibers in tissues, in order to identify the antigenic components. The extracts were also tested with antisera to several matrix proteins that have been reported to be associated with zonular fibers. Proteins reactive with antisera to bovine serum albumin, serum Immunoglobulins and fibronectin were present. No bands reactive with antisera to a-elastin, prealbumin, amyloid P component, collagen VI, lysyl oxidase or monoclonal antibody to fibrillin were demonstrated. The major nonserum protein band identified by both antisera was a 32kD polypeptide. An equally strong 250kD polypeptide was shown by the antiserum to guanidine-dithiothreitol extracted zonular fibers. Both of these proteins were PAS-positive and were demonstrated also by the antisera in extracts of bovine elastic neck ligament. Whether the two glycoproteins are related to each other, with the higher molecular weight protein either a precursor or aggregate form, is not yet clear. They appear to bear a close relationship to the elusive core microfibrillar protein.