Analysis of spontaneous hybridomas generated from nonobese diabetic (NOD) mice indicates that the natural autoantibody repertoire of NOD mice is highly active compared with C57BL/6 and BALB/c mice. This property of increased B cell activity is present early in life (4 wk) and persists in older mice of both sexes. Even when selected for binding to a prototypic β cell Ag, such as insulin, NOD mAb have characteristics of natural autoantibodies that include low avidity and broad specificity for multiple Ags. Analyses of the variable region of Ig H chain (VH) and variable region κ L chain genes expressed by six insulin binding mAb show that V gene segments are often germline encoded and are identical with those used by autoantibodies, especially anti-dsDNA, from systemic autoimmune disease in MRL, NZB/W, and motheaten mice. VH genes used by four mAb are derived from the large J558 family and two mAb use VH7183 and VHQ52 genes. The third complementarity-determining region of Ig H chain of these mAb have limited N segment diversity, and some mAb contain DNA segments indicative of gene replacement. Genetic abnormalities in the regulation of self-reactive B cells may be a feature that is shared between NOD and conventional systemic autoimmune disorders. In NOD, the large pool of self-reactive B cells may fuel autoimmune β cell destruction by facilitating T-B cell interactions, as evidenced by the identification of one mAb that has undergone Ag-driven somatic hypermutation.