No clear guidelines for indirect immunofluorescence (IIF) detection and interpretation of antineutrophil cytoplasmic antibodies (ANCA) have been proposed for inflammatory bowel diseases (IBD). We evaluated the reliability of the combined use of ethanol- and formalin-fixed neutrophil substrates to identify atypical perinuclear ANCA (P-ANCA) by IIF under routine laboratory circumstances. A total of 204 IBD patients were assessed with four different fluorescent substrates in two distinct laboratories. Antibodies against myeloperoxidase, proteinase-3, and other specific granule proteins (elastase, lactoferrin, cathepsin G, lysozyme, and bactericidal permeability-increasing protein) were measured by an enzyme-linked immunosorbent assay. The combined application of ethanol- and formalin-fixed slides to detect atypical P-ANCA resulted in a lack of agreement between assays (kappa, < or =0.39) in the interassay study and moderate agreement in the interobserver study (kappa, 0.42). After atypical and typical P-ANCA patterns were combined, the consensus improved greatly. A total of 26.9% of patients were P-ANCA positive by at least two tests (44.3% of ulcerative colitis [UC] and 13.1% of Crohn's disease [CD] patients; P < 0.0001), while overall ANCA positivity was 22.5% to 34.8%. The combined application of ethanol-fixed and formaldehyde-fixed neutrophil substrates did not facilitate differentiation between P-ANCA and atypical P-ANCA, and the results were not consistent when substrates from different sources were used. Combining all P-ANCA ensures the highest sensitivity and specificity in differentiating UC from CD.