We present an infant with severe familial hyperinsulinism in whom glucose production rate, lipolysis, and gluconeogenesis from glycerol were measured by use of glucose and glycerol labelled with stable isotopes. Administration of a single dose of glucagon (0.1 mg/kg) caused an increase in glucose production rate by near 140% from 4.2 to 10.1 mg·kg<sup>–1</sup>·min<sup>–1</sup>. The rate of appearance of glycerol, reflecting the rate of lipolysis, decreased from 15.1 to 12.6 µmol· kg<sup>–1</sup>·min<sup>–1</sup>. The amount of glycerol converted to glucose by gluconeogenesis was 9.1 µmol·kg<sup>–1</sup>·min<sup>–1</sup> before and 10.5 µmol·kg<sup>–1</sup>·min<sup>–1</sup> after glucagon administration. We conclude that the marked rise in glucose production rate was mainly the result of increased glycogenolysis. Following the trial, the child was started on long-acting (zinc-protamine) glucagon which made it possible to discontinue intravenous treatment with glucose.