This study compared the performance of a flow cytometric immunoassay for antineutrophil cytoplasmic antibodies (ANCA) directed against proteinase 3 (PR3) and myeloperoxidase (MPO), with indirect immunofluorescence (IIF) and ELISAs from 12 different manufacturers. Sera were from patients with active (n=55) or treated (n=68) small vessel vasculitis, or inflammatory bowel disease (IBD, n=22). The immunoassay specificity was 88% compared with 96% for IIF and 94% (median, range 91-96%) for both ELISAs. Its sensitivity in treated disease was 82% compared with 84% for IIF and 69% (median, range 57-82%) for the ELISAs. The immunoassay's specificity was 88% which was the same as the median for both ELISAs (range 84-95%). The PR3- and MPO-ANCA immunoassay was almost as sensitive as IIF, and more sensitive than, but just as specific as, most ELISAs, in detecting ANCA in active and treated vasculitis. A major advantage of this assay is its ability to be further modified to simultaneously screen for a panel of autoantibodies relevant to vasculitis.