We used a newly developed immunochemiluminometric assay of proinsulin to determine its relative utility vis-a-vis C-peptide and insulin for the diagnosis of insulinoma. The evaluation was conducted in 20 consecutive patients with histologically confirmed insulinoma and 22 normal subjects who underwent a prolonged fast according to a standard protocol. Patients with insulinoma fasted to the point of demonstrating Whipple's triad; normal subjects fasted to 72 h. At the end of the prolonged fast, when the glucose value was 2.8 mmol/L or less (50 mg/dL), all three hormones had equal sensitivity (100%) in detecting insulinoma with no overlap with the values of normal subjects. When glucose levels were between 2.8 mmol/L (50 mg/dL) and 3.3 mmol/L (60 mg/dL) at the end of the prolonged fast, proinsulin was better than C-peptide and insulin in the diagnosis of insulinoma. The sensitivity was 90% for proinsulin and 85% for both C-peptide and insulin. Therefore, proinsulin not only is useful for the diagnosis of insulinoma, but it may have greater diagnostic accuracy than C-peptide and insulin.