Serological testing for anti-neutrophil cytoplasmic antibodies (ANCA) has become an important tool for supporting a diagnosis of systemic necrotizing small vessel vasculitis: Wegener's granulomatosis, microscopic polyangiitis, Churg-Strauss syndrome and oligo-symptomatic forms of these. These so-called ANCA-associated vasculitides most often necessitate the institution of therapies with cytotoxic as well as anti-inflammatory agents, and hence, a firmly established diagnosis is mandatory to avoid unnecessary and risky treatment. In the laboratory of today the most appropriate way to detect the presence of vasculitis-associated ANCA is by using both indirect immunofluorescence and direct enzyme immuno-assay for antibodies to proteinase 3 and myeloperoxidase. The diagnostic specificity of these latter assays towards systemic vasculitis can only be secured by setting a suitably high cut-off value, chosen in collaboration with clinicians after testing carefully selected disease control sera. When classical cytoplasmic ANCA as well as a significant level of proteinase 3-ANCA are found in a given serum this combined result strongly indicates vasculitis. Similarly, the combination of perinuclear ANCA and a significant level of myeloperoxidase-ANCA is close to 100% specific for vasculitis.