To study the heterogeneity of islet cell antibodies (ICA), recombinant rat and human GAD65 expressed as bacterial fusion proteins were used to inhibit ICA reactivity in sera from recent onset type 1 diabetic children and ICA-positive first degree relatives of diabetic patients. Rat GAD65 was expressed as a fusion protein in the expression vector RSET and inhibited ICA (GAD+ICA) in 26% of 23 recent onset patients, 29% of 14 ICA positive first degree relatives (FDR) who progressed to diabetes (prediabetics) and 50% of 20 FDR who did not progress to diabetes 18 months to 5 years (31 +/- 14 months) after collection of the sample. GAD+ICA were inversely associated with the presence of insulin autoantibodies (IAA) (P = 0.006). GAD antibodies (GAD-Ab) were also detected by immunoprecipitation of in vitro transcribed and translated [35S] methionine-labelled human GAD65. GAD-Ab were present in 83% of recent onset patients, 86% of prediabetics and 95% of the relatives who did not progress to diabetes. The level of GAD-Ab was higher in the presence of GAD+ICA (1.39 +/- 0.57 vs 0.79 +/- 0.6 index units; P = 0.001). ICA levels were higher in GAD-Ab negative than in GAD-Ab positive sera (377 +/- 256 vs 195 +/- 231 JDFU; P = 0.03). Our results confirm that a recombinant GAD65 fusion protein can be used to detect ICA heterogeneity. However, neither inhibition of ICA with recombinant GAD, nor direct detection of GAD-Ab improved the prediction of progression to clinical diabetes in ICA positive FDR.