Background: Autoantibodies for the 65-kDa form of glutamic acid decarboxylase (GAD65) and protein tyrosine phosphatase-like protein (IA-2) are measured for risk prediction and diagnosis of autoimmune diabetes mellitus. There is a lack of adequate nonisotopic alternatives to the most widely used method for both autoantibodies, which is a radiobinding assay (RBA). Methods: We compared two commercially available immunoassays, an ELISA and a time-resolved immunofluorometric assay (TR-IFMA), with RBA. Results: We found excellent agreement between the RBA and ELISA for measurement of GAD65 autoantibodies (GADAs); they showed comparable analytical precision in the cutoff range and achieved similar diagnostic specificity. The ELISA identified more GADA-positive individuals among patients with new-onset type 1 diabetes than did the RBA [89% (95% confidence interval, 78–95%), vs 71% (58–82%); P Conclusions: The GADA ELISA and IA-2A TR-IFMA behave comparably with RBA and are thus suitable for use in the clinical laboratory.