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Aims: Glutamate decarboxylase (GAD) antibodies are the most widely used predictive marker for Type 1 diabetes, but many individuals currently found to be GAD antibody-positive are unlikely to develop diabetes. We have shown previously that radioimmunoassays using N-terminally truncated 35S-GAD65(96–585) offer better disease specificity with similar sensitivity to full-length 35S-GAD65(1–585). To determine whether assay performance could be improved further, we evaluated a more radically truncated 35S-GAD65(143–585) radiolabel. Methods: Samples from people with recent-onset Type 1 diabetes (n = 157) and their first-degree relatives (n = 745) from the Bart's–Oxford family study of childhood diabetes were measured for GAD antibodies using 35S-labelled GAD65(143–585). These were screened previously using a local radioimmunoassay with 35S-GAD65(1–585). A subset was also tested by enzyme-linked immunosorbent assay (ELISA), which performs well in international workshops, but requires 10 times more serum. Results were compared with GAD antibody measurements using 35S-GAD65(1–585) and 35S-GAD65(96–585). Results: Sensitivity of GAD antibody measurement was maintained using 35S-GAD65(143–585) compared with 35S-GAD65(1–585) and 35S-GAD65(96–585). Specificity for Type 1 diabetes was improved compared with 35S-GAD65(1–585), but was similar to 35S-GAD65(96–585). Relatives found to be GAD antibody-positive using these truncated labels were at increased risk of diabetes progression within 15 years, compared with those positive for GAD(1–585) antibody only, and at similar risk to those found GAD antibody-positive by ELISA. Conclusions: The first 142 amino acids of GAD65 do not contribute to epitopes recognized by Type 1 diabetes-associated GAD antibodies. Low-volume radioimmunoassays using N-terminally truncated 35S-GAD65 are more specific than those using full-length GAD65 and offer practical alternatives to the GAD antibody ELISA for identifying children at increased risk of Type 1 diabetes.