The antimitochondrial response in primary biliary cirrhosis (PBC) is the most highly directed and specific self-reacting antibody in human immunopathology. Originally, antimitochondrial antibodies (AMAs) were detected by indirect immunofluorescence (IIF) and found in approximately 90% of well-documented patients with PBC. The introduction of recombinant autoantigens and the use of immunoblotting have increased the sensitivity and specificity of AMAs, and they are now considered positive in approximately 95% of patients with PBC. Clearly, accurate autoantibody detection represents one of the fundamental requirements for reliable diagnostics in autoimmunity. To address the 5% of AMA-negative patients with PBC, we have generated and validated a bead assay for the detection of AMA. We enrolled 120 patients with PBC, including a non-random group of 30 rigorously proven AMA-negative patients, 50 healthy subjects, and 74 controls with autoimmune diseases ( T he presence of circulating anti-mitochondrial antibodies (AMAs) represents a key diagnostic feature of primary biliary cirrhosis (PBC). Using highly sensitive assays with recombinant antigens, up to 95% of PBC patients have sera autoantibodies directed against the ubiquitous 2-oxo-acid-dehydrogenase complexes involved in mitochondrial energy metabolism. AMA positivity with its unique prevalence rate among autoimmune diseases 1 represents one of the three diagnostic criteria for PBC, the others being liver histology compatible with the disease and a persistent elevation of alkaline phosphatase. 2 Approximately 5% of well-documented PBC patients do not react with any of the mitochondrial antigens using currently available assays. Over the past decade, the "AMA-negative" population has been progressively reduced in our laboratory by the development of more sensitive detection techniques. 3 Nonetheless, the persistence of some AMA-negative subjects raises the question of whether these patients represent a subpopulation with distinct clinical features or simply have antibody titers and specificities undetectable with available technology. 4 Recent technological advances have led to the development of increasingly automated detection methods associated with more rapid, accurate, and reliable assaying, 5 which could address the question. The From the