OBJECTIVE In a previous investigation into corneal autoimmunity, it was demonstrated that a putative autoantigen, a protein of 66 kDa, present in bovine corneal epithelium, binds circulating autoantibodies in approximately 60% of patients with Wegener's granulomatosis (WG). The aim of the present study was to characterize and identify the 66-kDa protein. METHODS A purification protocol was established for the 66-kDa protein using standard chromatography techniques. During the purification procedure it became clear that the 66-kDa protein detected in patients' sera was in fact two proteins, both running at 66 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, that eluted in different fractions on DE-52 chromatography columns. These two proteins have been labeled bovine corneal epithelial antigen-A and -B (BCEA-A and BCEA-B). Further investigations of antibody binding have demonstrated that patients' sera bind to either one or the other of these proteins with no cross-reactivity between them. Separated BCEA-A and BCEA-B protein extracts were immunoblotted with 27 WG patients' sera, 10 Churg-Strauss syndrome (CSS) patients' sera, 31 rheumatoid arthritis (RA) patients' sera, and 40 healthy control subjects' sera from the blood bank. RESULTS Forty-six percent of WG patients' sera had antibodies to one of the 66-kDa antigens, whereas none of the healthy control subjects' sera had 66-kDa antibodies (P < 10(-5)). In the WG group, 31% were positive to BCEA-A (versus controls, P = 0.0023), and 15% were positive to BCEA-B. WG patients with peripheral ulcerative keratitis (PUK) had a significant association with anti-BCEA-A antibodies when compared with healthy control subjects (50%, P < 10(-6)). However, in the RA group with no eye disease there was an association with BCEA-A (25%, P = 0.011) but not in the RA group with PUK. The frequency of anti-BCEA-B antibodies was significantly increased in patients with CSS (60%, P < 10(-7)). CONCLUSIONS In summary, it has been shown that vasculitis patients have antibodies to two 66-kDa corneal antigens and that autoantibodies to these antigens are mutually exclusive. It has also been shown that antibodies to BCEA-B are associated with CSS, whereas BCEA-A antibodies are associated with WG and RA.