The goal of this study was to address whether antiislet autoantibodies appear sequentially or simultaneously before the onset of type I diabetes. We analyzed sequential serum samples from 155 siblings and offspring (aged < 7 yr) of patients with type I diabetes from the Denver Diabetes Autoimmunity Study in the Young study and from a separate group of first degree relatives (aged 2-40 yr) for autoantibodies reacting with three defined autoantigens: glutamic acid decarboxylase (GAD65), insulin, and ICA512/IA-2. The youngest age at which 1 of the 3 autoantibodies appeared was 1.1 yr, and the oldest was 60.9 yr. Of the total 26 autoantibody conversion events observed, in only 3 instances did more than 1 autoantibody appear simultaneously. Among individuals (n = 12) with sequential conversion to expression of multiple autoantibodies, anti-GAD65 autoantibodies or antiinsulin autoantibodies appeared first (4 expressed antiinsulin autoantibodies first, and 8 anti-GAD65 autoantibodies first). We conclude that antiislet autoantibodies usually appear sequentially and not simultaneously. This corroborates early suggestions that humoral autoimmunity to islets develops chronically in a process usually measured in months to years. As expression of multiple autoantibodies is associated with a high risk of progression to diabetes, and sequential appearance of autoantibodies can occur late in life, long term follow-up is necessary to fully delineate the relationship of diabetes risk to autoantibody expression.