To study glucose counterregulation under conditions approximating those of clinical disorders in which hypoglycemia develops gradually and is reversed over a prolonged period, we injected regular insulin subcutaneously, in a dose (0.15 U/kg) selected to produce two- to threefold increases in plasma insulin, in 11 normal human volunteers and measured plasma glucose, insulin, C-peptide, and counterregulatory hormone concentrations as well as rates of glucose production, glucose utilization, and insulin secretion over 12 h. The data suggest that the mechanisms of gradual recovery from prolonged hypoglycemia may differ from those of rapid recovery from short-term hypoglycemia produced by intravenous injection of insulin in that 1) both stimulation of glucose production and limitation of glucose utilization contribute to recovery from prolonged hypoglycemia; 2) increases in glucagon, epinephrine, growth hormone, and cortisol secretion as well as a decrease in insulin secretion may all participate in glucose counterregulation during prolonged hypoglycemia; 3) epinephrine may play a more important role than glucagon during prolonged hypoglycemia. The latter two conclusions are based primarily on the temporal relationships between changes in the rates of glucose turnover and changes in plasma hormone concentrations and should not be considered proved. However, they provide the basis for testable hypotheses concerning the physiology of gradual recovery from prolonged hypoglycemia that can be expected to be relevant to the pathophysiology of clinical hypoglycemia.