Anti-proteinase 3 antibodies are a subgroup of anti-neutrophil cytoplasmic antibodies (ANCA), and we have established an ELISA for their detection using high performance liquid chromatography (HPLC)-purified protein. This assay is sensitive and specific: inhibition studies have shown that despite the homology between proteinase 3 and elastase there is no cross-reactivity between the corresponding antibodies for their targets. Anti-proteinase 3 antibodies were associated most often with cytoplasmic fluorescence (17/22, 77%), but occasionally with a perinuclear (3/22, 14%) or atypical pattern (1/2). These antibodies were found in 23 out of 76 sera (30%) that were positive in an ELISA based on a crude neutrophil cytoplasmic extract, and they were associated with both 29 and 55 kD bands on Western blots. Anti-proteinase 3 antibodies were found in most individuals with active Wegener's granulomatosis (10/13, 77%), but less often in individuals with microscopic polyarteritis (2/10, 20%) or segmental necrotizing glomerulonephritis (3/6, 50%). However, anti-proteinase 3 antibodies were not detected in any of 32 sera from individuals with rheumatoid arthritis or systemic lupus erythematosus (SLE). Occasionally anti-proteinase 3 antibodies were associated with anti-glomerular basement membrane antibodies (1/11, 9%) or with anti-myeloperoxidase antibodies (1/11, 9%). IgM anti-proteinase 3 antibodies were uncommon (2/22 sera, 9%), and no IgA antibodies were demonstrated in any of 22 sera from patients with active systemic vasculitis. Significantly more individuals presented with anti-proteinase 3 antibodies in April-May-June, suggesting that an infective agent prevalent in Autumn might have a causative role in the associated diseases. Anti-proteinase 3 antibodies are the most common target antigen associated with Wegener's granulomatosis and cytoplasmic fluorescence.