BACKGROUND Autoantibodies to glutamic acid decarboxylase (GADAs), specifically the 65-kDa isoform GAD65, and autoantibodies to the protein tyrosine phosphatase-like molecule IA-2 (IA-2As) predict development of diabetes. Our aim was to develop a time-resolved immunofluorometric (TR-IFMA) dual-label assay method for the simultaneous detection of these autoantibodies and to evaluate the diagnostic sensitivity of the method compared with single-label TR-IFMA and fluid-phase radiobinding assay (RBA) in screening children with type 1 diabetes. METHODS We incubated combined biotinylated GAD65 and IA-2 proteins, glutathione S-transferase (GST)-IA-2, europium-labeled GAD65, terbium-labeled anti-GST antibody, and serum sample or calibrator and transferred aliquots to a streptavidin-coated 96-well microtiter plate for a second incubation. After washing, we added Delfia Enhancement solution to each well and measured the fluorescence of Eu. We developed the Tb fluorescence signal by use of the Delfia Enhancer solution and measured it. We analyzed serum samples from a cohort of 100 children with newly diagnosed type 1 diabetes. RESULTS The correlation coefficients between the autoantibody concentrations measured by dual- and single-label TR-IFMA assays were 0.962 for GADA and 0.874 for IA-2A. Among 100 children with newly diagnosed diabetes, 65 of them were GADA positive in the dual-label assay, 64 in the single-label assay, and 66 in the RBA GADA assay. Seventy-four of the children tested positive for IA-2A in both TR-IFMA assay types, and 79 in the RBA IA-2A assay. CONCLUSIONS The novel dual-label immunofluorometric assay performed comparably to the separate, single-label GADA and IA-2A assays in screening for beta-cell autoimmunity in children with newly diagnosed type 1 diabetes.