To assess the changes in counterregulatory hormones overnight after an afternoon of structured exercise or sedentary activity in children with type 1 diabetes mellitus (T1DM), the Diabetes Research in Children Network (DirecNet) studied 50 children (10 to <18 yr) with T1DM in five clinical research centers on two separate days (with and without an afternoon exercise session) using a crossover design. Glucose, epinephrine, norepinephrine, cortisol, growth hormone (GH), and glucagon concentrations were measured hourly overnight. Nocturnal hypoglycemia [plasma glucose concentrations < or =70 mg/dL (3.9 mmol/L)] occurred more frequently on the nights following exercise (56 vs. 36%; p = 0.008). Mean hourly concentrations of most hormones did not differ between sedentary or exercise nights or between nights with or without hypoglycemia. Spontaneous nocturnal hypoglycemia only stimulated small increases in plasma epinephrine and GH concentrations and failed to cause a rise in norepinephrine, cortisol, or glucagon levels in comparison with values during the hour before or after hypoglycemia or other times during those same nights. Counterregulatory hormone responses to spontaneous nocturnal hypoglycemia were markedly decreased regardless of whether there was antecedent afternoon exercise in children with T1DM. Sleep-induced impairments in counterregulatory hormone responses likely contribute to the increased risk of hypoglycemia during the entire overnight period in youth with T1DM.