Anti-neutrophil cytoplasmic autoantibodies (ANCA) react with antigens in the cytoplasm of neutrophils and monocytes, and are found in the blood of patients with necrotizing vasculitis and pauci-immune necrotizing and crescentic glomerulonephritis. The standard techniques for ANCA analysis use human polymorphonuclear leukocytes (PMN) as a source for antigen. A comparison was made between ANCA reactivity with human PMN and HL-60 cells. Fifty-five ANCA-positive and 17 ANCA-negative sera were reacted with HL-60 cells in an indirect immunofluorescence microscopy assay to assess the ability of HL-60 cells to distinguish between positive and negative reactivity and between the two major types of ANCA. The HL-60 cell method agreed with a PMN method in all but one instance. In an enzyme immunoassay, HL-60 primary granules could be used to detect ANCA. Evidence is also presented that the perinuclear staining of HL-60 cells and neutrophils by ANCA with anti-myeloperoxidase specificity is caused by the nucleophilic properties of myeloperoxidase. Reactivity with HL-60 cells further elucidates the antigen specificity of both types of ANCA and indicates that HL-60 cells are a suitable tissue culture cell line for investigating the pathobiology of ANCA.