OBJECTIVE To determine the antigens recognized by sera containing classic anti-neutrophil cytoplasmic antibodies (c-ANCAs) and perinuclear anti-neutrophil cytoplasmic antibodies (p-ANCAs). METHODS A total of 160 serum samples (all from a reference laboratory) that were originally collected from different clinics for ANCA tests were examined for c-ANCA and p-ANCA by indirect immunofluorescence (IIF). All positive sera were further tested for reactivity to proteinase 3 (PR3), myeloperoxidase (MPO), lactoferrin (LF), and lysozyme (LZ) by enzyme-linked immunosorbent assay (ELISA). In addition, sera from 110 patients with systemic lupus erythematosus (SLE), 51 patients with rheumatoid arthritis (RA), and 40 healthy subjects were also tested for reactivity to these antigens. RESULTS HF detected ANCA in 81 (51%) of the 160 clinical serum samples. Of these 81 serum samples, 21 (26%) contained c-ANCA and 60 (74%) contained p-ANCA. P-ANCA was more commonly found in antinuclear antibody (ANA)-positive sera than in ANA-negative sera (p < 0.01). Of the 21 serum samples positive for c-ANCA, 12 (57%) reacted to PR3, four (19%) to LF, four (19%) to LZ, and three (14%) to MPO on ELISA. By contrast, of the 60 sera positive for p-ANCA, 15 (25%) reacted to MPO, 13 (22%) to PR3, eight (13%) to LF, and four (7%) to LZ. The prevalence of ANCA specificities in serum samples from SLE patients were as follows: anti-PR3, 0%; anti-MPO, 1%; anti-LF, 27%; and anti-LZ, 29%. The prevalence of ANCA specificities in serum samples from RA patients were as follows: anti-PR3, 6%; anti-MPO, 2%; anti-LF, 8%; anti-LZ, 4%. CONCLUSIONS Sera positive for c-ANCA and p-ANCA reacted to diverse cytoplasmic antigens from neutrophils. P-ANCA was found in 55% of ANA-positive serum samples. LF and LZ were most commonly found in serum samples from patients with SLE.