Abstract The characteristics of humoral immunity have been proposed to reflect the bias between two T helper (Th) lymphocyte subsets: Th1 cells, which activate cell-mediated immunity, and Th2 cells, which mediate humoral immunity. The present study aimed to characterize the humoral immunity to beta-cell autoantigens insulin and glutamic acid decarboxylase (GAD65) in preclinical and clinical type 1 diabetes. Insulin antibodies were analyzed in pregnant women with or without type 1 diabetes and their newborn infants and in prediabetic children. Epitope or/and isotype-specific GAD65 antibodies (GAD65Abs) were analyzed in prediabetic children, in children and adolescents diagnosed with type 1 diabetes, and in patients with the autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) syndrome. Antibodies were determined by radioligand immunoassays. The humoral immune response to insulin and GAD65 was observed to be a highly dynamic process, comprising mainly the IgG1 subclass and, less frequently, other IgG subclasses. GAD65Abs were directed primarily to the middle region and secondarily to the C-terminal region of GAD65 as a consequence of epitope spreading. Young children who progressed to overt type 1 diabetes were characterized by a broad initial isotype response to insulin and GAD65 and by a strong IgG1 and IgG3 response to insulin. Children who did not progress to clinical type 1 diabetes were characterized by an emerging IgG4 response to GAD65. Rising levels of GAD65Abs targeted to the middle region of GAD65 were associated with high titers of islet cell antibodies and a decreased requirement for exogenous insulin, probably reflecting a persistent residual beta-cell mass, in patients with manifest type 1 diabetes. Non-immunoglobulin insulin-binding activity was observed to be induced by pregnancy. APECED-associated humoral autoimmunity to GAD65 did not differ markedly from that observed in subjects with type 1 diabetes alone. In conclusion, isotype-specific GAD65 and especially insulin antibodies are valuable markers of the risk of progression to type 1 diabetes in young children. The appearance of an initial IgG3 subclass response and a strong IgG3 response to insulin in children who progressed to overt type 1 diabetes may reflect the role of cytotoxic Th1-biased immunity in the disease process leading to clinical presentation of type 1 diabetes.