Glutamic acid decarboxylase (GAD) and tyrosine phosphatase IA-2 antibody levels were measured in 375 healthy children and adults and in 187 children with newly diagnosed type 1 diabetes mellitus (DM). GAD antibody levels were determined by radioimmunoassay, and IA-2 antibody levels by immunoprecipitation. Healthy children had higher GAD antibody levels than adults (p < 0.001). The 98th percentile was 1.60 U/ml in children and 1.16 U/ml in adults. IA-2 antibody levels did not differ between these two cohorts. Children with DM had higher GAD and IA-2 antibody levels than healthy children (p <0.001). Based on receiver operating characteristics (ROC) analysis, elevated levels of these antibodies showed high specificity rates (+/- SE) of 0.968 +/- 0.14 to 1.00 +/- 0.00. The sensitivity ranged between 0.439 +/- 0.037 (both antibodies elevated) and 0.850 +/- 0.027 (at least one antibody elevated). These data emphasize the importance of establishing age-specific reference values for DM-related antibodies in the background population before applying them for screening and intervention studies.