Anti-insulin autoantibodies were detected in NOD mice using ELISA. The antibodies were detected as early as at 5 weeks of age, long before onset of clinically overt diabetes, especially in diabetes-prone female mice. The anti-insulin specificity was verified by passage on affinity chromatography insulin columns and demonstration that the anti-insulin activity was located on the F (ab') 2 region of the immunoglobulins. The presence of anti-insulin antibodies in prediabetic NOD mice provides a unique possibility for studying their significance and their eventual pathogenic role in the development of insulin-dependent diabetes.