In order to investigate the causes underlying metabolic instability in type I diabetes mellitus, we studied 8 unstable (group 1) and 4 well-controlled (group 2) diabetic patients, matched for age and duration of diabetes. Subjects were connected overnight to an artificial pancreas and brought to normoglycemia. On the following morning, insulin administration was discontinued for 6 hours and both metabolic and hormonal studies were carried out during this period. After insulin withdrawal, group 1 showed a faster rise of blood glucose (peak: 324.63±24.93vs 175.25±42.63 mg/dl, p<0.01), β-OH-butyrate (peak: 2,273.25±415.78vs 550.50±158.17 µmol/1, p<0.01), and glycerol (164.10±38.90vs 28.25±10.6 µmol/1, p<0.01). C-peptide secretion increased in group 2 from 0.09±0.052 to 0.22±0.099 pmol/ml whereas it remained almost undetectable in group I (p<0.01, group 1vs group 2). Growth hormone, cortisol and immunoreactive glucagon were not significantly different in the two groups at any time after insulin withdrawal. Free insulin, after repeated s.c. or i.m. injection of porcine monocomponent insulin (10 IU), was not different in the two groups. We concluded that type I diabetic patients showing severe metabolic instability produced more glucose, ketone bodies and glycerol after insulin withdrawal than control ‘stable’ patients. This difference could not be accounted for by an excessive secretion of counterregulatory hormones or by an erratic insulin absorption from the injection sites and may have been related to the degree of B-cell failure, as measured by the absence of C-peptide and/or to the degree of insulin resistance.