We observed 42 initially non-diabetic siblings of affected children to characterize the humoral immune response to the 65 kDa isoform of glutamic acid decarboxylase (GAD65) in preclinical type I diabetes. During the observation period with a mean duration of 9.6 years 21 siblings progressed to type I diabetes. The humoral immune response to GAD65 was observed initially as a simultaneous response to the middle (M) and carboxy (C)-terminal regions of the GAD65 molecule in most cases, and if the response was restricted initially to the middle region, it spread rapidly to the C-terminal domain and in a few cases later to the amino (N)-terminal domain. There was some heterogeneity in the GAD65 isotype response, but it was composed mainly of antibodies of immunoglobulin (Ig) G1 subclass. Responses of IgG2-, IgG4-, IgM- and IgA-GAD65Ab were observed frequently, whereas IgE- and IgG3-GAD65Ab responses were seen more rarely. Initially, the non-progressors tended more often to have IgG2- and IgG4-GAD65Ab than the progressors. As a sign of a dynamic process a significant isotype spreading was seen for IgG2-GAD65Ab (P < 0.05) and close to significant for IgM (P = 0.06) among progressors and for IgM-GAD65Ab (P < 0.05) among non-progressors during the observation period. This study failed to identify any GAD65 epitope- or isotype-specific antibody reactivity that could be used as a marker for progression to disease, as such progression was not associated with any specific changes in reactivity over time. Our findings indicate that epitope- and isotype-specific GAD65 antibodies are hardly capable of separating progressors from non-progressors among GAD65Ab-positive first-degree relatives of children with type I diabetes.