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We propose a four step flow-chart to define ANCA positivity and antigenic target. 1st step: indirect immunofluorescence test on ethanol-fixed human granulocytes (IIF-E), as screening test. Different staining patterns can be observed: a granular cytoplasmic fluorescence (C-ANCA), a smooth or fine granular perinuclear fluorescence (P-ANCA) and an intermediate pattern (X-ANCA). Antinuclear antibodies (ANA) may mimic P-ANCA. 2nd step: IIF test on formalin-fixed human granulocytes (IIF-F) differentiates true P-ANCA from ANA: most of P-ANCA show cytoplasmic pattern, whereas ANA are negative. 3rd step: IIF test on monkey liver sections (IIF-M) investigates simultaneous ANA and P-ANCA positiveness. P-ANCA positive sera show an exclusive reactivity with neutrophils infiltrating the portal tract, whereas ANA react with hepatocytes nuclei. 4th step: to characterize antigenic target, a solid phase assay, using purified proteins as substrates, is performed. We found 17 C-ANCA (6 PR3, 3 MPO, 1 Lys, 1 Cat G and 6 unknown antigens) out of 173 patients screened with IIF-E. 21 P-ANCA positive sera have been investigated by IIF-F test: 15 showed a cytoplasmic pattern; EIA test gave the following results: 6 MPO, 2 LF, 5 unknown antigens; 2 cases were positive for two antigens, MPOLF. Using IIF-M on the 6 IIF-F negative sera, we observed: 2 false positives (ANCA-/ANA-), one ANCA+/ANA+ (antigen LF), 3 ANCA+/ANA- (unknown antigens). The flow chart suggested allows to analyse in detail ANCA, using easily available commercial kits.