In October 2000, the First Murine Autoantibody Workshop was held as part of an International Workshop on Lessons from Animal Models for Human Type 1 Diabetes. This first workshop identified insulin, but not glutamic acid decarboxylase (GAD) or IA-2, as specific autoantigens of humoral immunity in nonobese diabetic (NOD) mice. The goals of the Second Murine Autoantibody Workshop, part of the Sixth Annual Meeting of the IDS, were to increase the number of participating investigators, attempt standardization of insulin autoantibody (IAA) results across laboratories, identify serologic evidence of humoral immunity to other beta cell antigens, and allow for validation of ELISA assays for autoantibody detection in NOD mice. Sixty-three coded samples (26 pooled NOD sera, 23 pooled C57BL/6 sera, and 14 diluted samples of an anti-insulin monoclonal antibody) were distributed to 12 participating laboratories. This second workshop demonstrated that, for nearly all laboratories, IAA measured by radioimmunoassay (RIA) provided a sensitive and specific assay capable of distinguishing diabetes-prone from nondiabetes-prone mice. Analyses involving the serially diluted anti-insulin monoclonal antibody offered hope that a standard reference unit for reactivity could be established. Surprisingly, two ELISA assays for IAA detection proved remarkably sensitive (i.e., 65% and 92%). However, subsequent absorption studies performed after the workshop (presented at the IDS meeting) brought into question whether ELISA assays for IAA do, in reality, detect anti-insulin immunities and whether assays for GAD and IA-2 autoantibodies distinguish diabetes-prone from nondiabetes-prone mice. In sum, this workshop continued to support the notion that IAA, as determined by RIA, could provide a sensitive and specific marker of anti-beta cell immunity in NOD mice.