In an attempt to clarify whether circulating insulin per se exerts an inhibitory effect on the hormonal responses to hypoglycemia, with special emphasis on glucagon secretion, nine healthy volunteers were exposed to low dose (244 pmol/kg.h) and high dose (1034 pmol/kg.h) iv insulin infusions for 3 h on two separate occasions. A close to identical arterial hypoglycemia of about 3.4 mmo/L was obtained in both tests by glucose clamping during the high dose test. The corresponding glucose concentration in the venous blood was significantly lower in the high dose test (2.5 +/- 0.1 vs. 3.0 +/- 0.1 mmol/L; P less than 0.01), while the plasma free insulin level was 4 times higher in the high dose test (897 +/- 50 vs. 208 +/- 14 pmol/L). Plasma glucagon was elevated in both experiments, but its rise was reduced during the high dose test after 1 h, yielding an incremental area under the glucagon curve that was significantly smaller than that obtained during the low dose test (213 +/- 70 vs. 348 +/- 81 ng/L.h; P less than 0.05). The plasma adrenaline, noradrenaline, GH, C-peptide, pancreatic polypeptide, and somatostatin profiles were similar in the two tests. We conclude that an inhibitory effect of circulating insulin on the glucagon response to hypoglycemia can be demonstrated in normal man during an infusion of insulin yielding a plasma concentration of about 900 pmol/L. The responses of other hormones studied are not significantly influenced by the circulating insulin level.