The serum levels of 34K insulin-like growth factor (IGF)-binding protein were measured by RIA in 88 type 1 diabetic patients, 9 patients with type 2 diabetes, 7 patients with insulinoma, 19 normal subjects (all after an overnight fast), and 82 normal subjects after a breakfast meal. In addition, the effect of 2- to 3-h euglycemic steady state hyperinsulinemia on serum IGF-binding protein and IGF-1 levels was studied in some subjects in each of the fasted groups. Compared with normal subjects, the mean serum IGF-binding protein levels were 4-fold (P < 0.001) higher in type I diabetic patients treated with conventional insulin injections, 2.5-fold (P < 0.001) higher in those treated with continuous sc insulin infusion, and 2-fold (P < 0.05) higher in patients with type 2 diabetes. In the patients with insulinoma, the mean IGF-binding protein level was 63% below normal (P < 0.001), and it normalized after removal of the tumor. There was a slight negative correlation between the IGF-binding protein level and insulin dose in the diabetic patients (r = −0.22; P < 0.05). In normal subjects, serum insulin concentrations were 2-fold higher (P < 0.001) and the IGFbinding protein level was 29% lower after a meal (P < 0.05) than in the fasting state. Serum IGF-I concentrations were virtually identical in the type 1 and 2 diabetic patients, insulinoma patients, and normal subjects. During steady state euglycemic hyperinsulinemia, the IGF-binding protein level fell by 40–70% in each group (P < 0.001), whereas the IGF-I level declined (P < 0.05) in the type 2 diabetic patients only. The decline of binding protein was closely related to the baseline level (r = 0.94; P < 0.001). No correlation was found between serum IGF- 1 and binding protein levels in any group. In conclusion, 1) serum 34K IGF-binding protein levels are elevated in type 1 and 2 diabetic patients and decreased in patients with insulinoma; 2) the serum binding protein, but not IGF-I concentration is decreased by acute hyperinsulinemia; and 3) these data suggest that the serum insulin concentration plays a role in regulation of the serum 34K IGF-binding protein concentration.