Autoantibodies against neutrophil granulocytes are frequently observed in patients with ulcerative colitis, but a precise description of the autoantigens involved is lacking. Therefore, sera from 75 patients with ulcerative colitis were studied for antibody specificities by means of indirect immunofluorescence microscopy, enzyme-linked immunosorbent assays, and immunoblotting of extracts of neutrophils, neutrophil granules and lymphocytes. Fifty-six percent of the sera reacted in indirect immunofluorescence with ethanol-fixed neutrophils. On formalin-fixed cells most sera shifted fluorescence pattern, indicating a cytoplasmic origin of antigen(s). Only a few sera reacted with specific, known antigens. Immunoblots showed reactions with a broad panel of antigens with preferences for proteins around 55-65 kDa, which were present in azurophilic granules and in cytosol, an 80-kDa protein found in the specific granules, and a 110-kDa unknown cytosol component. In conclusion, neutrophil-specific IgG autoantibodies from ulcerative colitis patients react with several different antigens, most of which are of nonnuclear origin.