Type 1 diabetes is an autoimmune disease characterized by immune-mediated destruction of the pancreatic beta cells. Insulin autoantibodies (IAAs) develop in many subjects at high risk for developing type 1 diabetes prior to onset of clinical disease and exposure to exogenous insulin, whereas insulin antibodies (IAs) commonly develop in patients treated with exogenous insulin. To investigate whether the binding characteristics of IAA and IA are similar, we measured eight different binding characteristics of IAAs from 19 insulin-naive first-degree relatives of type 1 diabetes patients and compared these to the binding characteristics of IAs from 19 type 1 diabetes patients treated with exogenous insulin. IAA and IA samples were matched for percentage insulin binding. Scatchard analysis revealed that IAAs have a two-slope representation similar to IAs-that is, two populations of antibodies, a high-affinity low-capacity population and a low-affinity high-capacity population. Binding properties of the two respective populations were found to be similar for IAAs and IAs. Sipps' equation was used to generate a Hill plot and produce an index of heterogeneity, which showed further similarity between IAAs and IAs. These results suggest that the IAAs found in subjects at increased risk for type 1 diabetes, like IAs, are likely to be polyclonal in nature. The similarities between IAAs and IAs support the hypothesis that both are induced by insulin presentation to the immune system.