Tests that positively identify individuals with ulcerative colitis, distinguishing them from patients with Crohn disease or other causes of colitis, have not been reliable. Genetic predisposition to inflammatory bowel diseases and genetic influence on immune regulation resulted in the clinical evaluation of potential serologic markers. In adults the presence of anti-neutrophil cytoplasmic antibody (ANCA) in serum identifies patients with ulcerative colitis. In this study we demonstrated that high levels of ANCA are present in 83% of children and adolescents with ulcerative colitis. Furthermore, the majority of patients with ulcerative colitis had a perinuclear pattern of these antibodies by indirect immunofluorescence. The combination of a positive ANCA and perinuclear indirect immunofluorescence pattern was 97% specific for ulcerative colitis. We conclude that determination of ANCA is a sensitive and specific clinical test for identification of children and adolescents with ulcerative colitis.