The cause of of ketotic hypoglycemia, the commonest form of hypoglycemia in childhood, is not known. The present study was undertaken to determine whether the primary defect in this condition is a deficiency of gluconeogenic precursor(s) or an abnormality in the hepatic gluconeogenic enzyme system. Plasma glucose, alanine, and insulin and blood beta-hydroxybutyrate (beta-OHB), pyruvate, and lactate levels were determined in eight ketotic hypoglycemic children and seven agematched controls maintained on a normal diet and after being fed a provocative hypocaloric low-carbohydrate diet (1200 kcal/1.73 m(2), 15% carbohydrate, 17% protein, and 68% fat). On a normal diet, overnight fasting plasma alanine (211+/-10 muM) and glucose (68+/-4 mg/100 ml) were significantly lower and blood beta-OHB (1.22+/-0.37 mM) significantly higher in ketotic hypoglycemic children than in controls (alanine, 315+/-15 muM; glucose, 81+/-3 mg/100 ml; beta-OHB, 0.18+/-0.08 mM). All ketotic hypoglycemic children developed symptomatic hypoglycemia (33+/-3 mg/100 ml) and ketosis (beta-OHB, 3.70+/-0.32 mM) 8-16 hr after starting the provocative diet and these changes were associated with a further decline in plasma alanine (155+/-17 muM). Normal children, even after 36 hr on this diet, maintained higher plasma glucose (48+/-2 mg/100 ml) and alanine (225+/-5 muM) and lower beta-OHB levels (2.56+/-0.44 mM). Intravenous infusions of alanine (250 mg/kg) uniformly restored the hypoglycemic plasma glucose levels of ketotic hypoglycemic children to normal. Cortisone acetate (300 mg/m(2)), given orally in three divided doses during feeding of the provocative diet, produced a 3- to 4-fold increase in plasma alanine within 4-6 hr after beginning steroid therapy and completely prevented the development of hypoglycemia and ketosis. Quantitative amino acid profiles were performed and demonstrated that alanine was the only gluconeogenic amino acid which differed significantly between the two groups. Plasma insulin and blood lactate and pyruvate levels did not differ significantly between normal and ketotic hypoglycemic children under all conditions examined. These results support the hypothesis that a deficiency in gluconeogenic precursor (e.g., alanine) rather than a defect in the hepatic gluconeogenic enzyme apparatus represents the most likely factor in the pathogenesis of ketotic hypoglycemia.