Antineutrophil cytoplasmic antibodies (ANCA) are a group of autoantibodies first associated with Wegener's granulomatosis (WG). In autoimmune liver diseases, ANCA have been described recently in patients with primary biliary cirrhosis (PBC), autoimmune hepatitis (AIH) and primary sclerosing cholangitis (PSC). Controversy exists about the prevalence and specificity of ANCA in patients with autoimmune liver diseases. The purpose of this study was first to assess the prevalence of antineutrophil antibodies in patients with primary biliary cirrhosis and second to identify possible target antigens of antineutrophil antibodies. Sera from 33 patients with PBC, 75 patients with AIH, 16 patients with PSC, 90 control sera (chronic hepatitis B, chronic hepatitis C, alcoholic liver cirrhosis, systemic lupus erythematosus) and sera from healthy blood donors were enrolled in the present study. ANA and ANCA were detected using standard protocols, antibodies against the nuclear antigen SP100 were detected by ELISA with recombinant antigen, antibodies against neutrophil alpha-granules were detected by ELISA. P-ANCA were found in two of 33 PBC sera, both patients presented with PBC/AIH overlap syndrome. In six of 33 (18%) PBC sera a nuclear dot like immunofluorescence pattern (antineutrophil nuclear antibodies, ANNA) was observed. These dots were small in size and located all over the nucleus, the anti-SP100 fluorescence. This result was confirmed by IFT on HEp-2 cells and by ELISA. High tier antibodies against SP100 were found to be specific for patients with PBC. Comparing the IFT results on HEp-2 cells and human neutrophils, we found an excellent correlation (Spearman's rank correlation coefficient = 0.968, P < 0.001). ANCA were detected in AIH type-1 (75%), SLA-positive AIH (36%) and PSC (63%). AIH type-2 was found to be ANCA negative. All PSC contained no antibodies against neutrophil alpha-granules. Our results show: 1. P-ANCA in patients with PBC indicate a PBC/AIH overlap syndrome; 2. ANNA in patients with PBC are mostly directed against SP100; 3. neutrophil granule components are not the ANCA specific antigens in patients with PSC.