The hyperinsulinism/hyperammonemia (HI/HA) syndrome is a form of congenital hyperinsulinism in which affected children have recurrent symptomatic hypoglycemia together with asymptematic, persistent elevations of plasma ammonium levels. We have shown that the disorder is caused by dominant mutations of the mitochondrial enzyme, glutamate dehydrogenase (GDH), that impair sensitivity to the allosteric inhibitor, GTP. In 65 HI/HA probands screened for GDH mutations, we identified 19 (29%) who had mutations in a new domain, encoded by exons 6 and 7. Six new mutations were found: Ser217Cys, Arg221Cys, Arg265Thr, Tyr266Cys, Arg269Cys, and Arg269His. In all five mutations tested, lymphoblast GDH showed reduced sensitivity to allosteric inhibition by GTP (IC50, 60-250 vs. 20-50 nmol/L in normal subjects), consistent with a gain of enzyme function. Studies of ATP allosteric effects on GDH showed a triphasic response with a decrease in high affinity inhibition of enzyme activity in HI/HA lymphoblasts. All of the residues altered by exons 6 and 7 HI/HA mutations lie in the GTP-binding domain of the enzyme. These data confirm the importance of allosteric regulation of GDH as a control site for amino acid-stimulated insulin secretion and indicate that the GTP-binding site is essential for regulation of GDH activity by both GTP and ATP.