C-Peptide, a marker for insulin secretion, is purported to be elevated in patients with insulinoma but diagnostic criteria have not been established. Thirty-seven patients with histologically confirmed insulinoma studied preoperatively, 19 normal subjects, and 2 patients who subsequently acknowledged self-administration of insulin underwent the prolonged fast (less-than-or-equal-to 72 h) according to a standard protocol. Plasma glucose, C-peptide, and insulin were measured every 6 h until plasma glucose was less than or equal to 3.3 mmol, then hourly until Whipple's triad was demonstrated or until 72 h without symptoms was reached. At the termination of the fasts, plasma was analyzed for sulfonylurea. Statistical analysis was by rank sum test. Data are expressed as median (range). The durations of fasts were 20 (2.5-68) h for patients with insulinomas and 72 h for normal subjects. At the end of fasts plasma glucose, C-peptide, and insulin concentrations were 2.2 (1.4-2.9) vs. 3.6 (2.7-5.5) mmol, P < 0.001; 0.60 (0.20-1.92) vs. 0.13 (0.07-0.43) nmol, P < 0.001; and 126 (35-840) us. 35 (35-126) pmol, P < 0.001, respectively, for insulinoma patients and normal subjects. All plasma samples were negative for sulfonylurea. Insulinoma patients had C-peptide values at the end of the fasts greater than or equal to 0.20 nmol whereas normal subjects and patients with insulin factitial hypoglycemia had C-peptide concentrations less than or equal to 0.10 nmol when plasma glucose was less than or equal to 2.8 mmol. Insulinoma is confirmed in a sulfonylurea negative patient with Whipple's triad during the prolonged fast and a concomitant C-peptide concentration greater than or equal to 0.20 nmol.