The AIRE gene (autoimmune regulator) has been identified as an important mediator of central tolerance against ectopically expressed peripheral antigens. Mutatations of AIRE are responsible for autoimmune polyendocrinopathy syndrome type I (APSI) in human characterized by a multiorgan autoimmune disease. Autoimmune hepatitis (AIH) is part of the clinical spectrum in humans and mice. 19% of patients with AIRE mutations develop an autoimmune hepatitis which is characterized by autoantibodies against cytochrome P450 2A6 and 1A2. We investigated AIH in mouse models with various AIRE mutations on different genetic backgrounds. Initially we characterized the immunohistochemical staining pattern of sera from AIRE−/− mice on rat liver, kidney and stomach sections and on HepG2 cells. In parallel, sera analysis has been done on western-blots loaded with whole liver lysate. We could demonstrate that the strength and broadness of the humoral immune response correlates well with disease activity of the AIH in AIRE deficient mice. We could show that AIH development is dependent on the genetic background used. Furthermore within the Balb/c background AIH development is dependent on the underlying AIRE mutation. Humoral immune responses in AIRE deficient mice are not directed against the antigens described for human AIH. We therefore set out to characterize the target antigens of the adaptive immune response in mice with AIH to study the defects in their immune tolerance. We therefore developed a column retention assay of for non-denatured liver antigens followed by a proteomics analysis with mass spectroscopy. With this approach we identified several potential new autoantigens of the humoral immune response.