Prevalence and clinical relevance of antibodies to soluble liver antigen (tRNP(Ser)Sec/SLA) in autoimmune hepatitis (AIH) have been investigated using partially purified or prokaryotically expressed antigen. The aim of this study was to improve the detection of anti-tRNP(Ser)Sec/SLA by establishing an immunoassay that was able to identify antibodies directed to conformational epitopes and to investigate the clinical implication of this autoantibody in autoimmune liver disease. By using eukaryotically expressed tRNP(Ser)Sec/SLA as target in a radioligand assay (RLA), 81 patients with autoimmune liver disease (AILD) (33 type 1 AIH, 31 type 2 AIH, and 17 autoimmune sclerosing cholantitis [ASC]), 147 pathologic, and 56 healthy controls were investigated. RLA results were compared with those obtained using a commercial enzyme-linked immunosorbent assay (ELISA) and immunoblot. Reactivity to tRNP(Ser)Sec/SLA was present in 58% of patients with type 1 and type 2 AIH, 41% with ASC, but in only 3 pathologic controls. RLA was similarly disease-specific but remarkably more sensitive than ELISA and immunoblot. A prospective study showed that anti-tRNP(Ser)Sec/SLA–positive patients run a severe clinical course, having worse histology, needing longer to achieve remission, relapsing and requiring liver transplantation or dying more frequently than anti-tRNP(Ser)Sec/SLA negative patients. Anti-tRNP(Ser)Sec/SLA production was favored by the possession of DR3 and A1-B8-DR3 in AIH type 1 and ASC, and prevented by the possession of A2 in all 3 types of AILD, particularly in type 2 AIH. In conclusion, anticonformational tRNP(Ser)Sec/SLA reactivity is frequent in type 1 and type 2 AIH and ASC, defining patients with a worse prognosis.