Lys(B28)Pro(B29) human insulin analogue (Lispro) is a newly developed monomeric insulin analogue with a rapid onset and short duration of action. The aim of the study was to compare the thresholds for the counterregulatory responses during a stepwise euglycaemic/hypoglycaemic clamp for insulin lispro (LP), human (H), and porcine (P) insulin in a randomized order in 12 healthy male volunteers (age 22.4 +/- 1.7 years, BMI 21.9 +/- 1.7 kg m-2). A euglycaemic period of 2 h was followed by three hypoglycaemic levels of 60 min each; from 150-210 min at 3.5 mmol l-1, 240-300 min at 3.0 mmol l-1 and 330-390 min at 2.5 mmol l-1. Plasma insulin levels during the 50 mU kg-1 h-1 infusions and blood glucose levels were not significantly different. The glucose requirements (mean +/- SD) during the last part of the euglycaemic period (90-120 min) tended to be higher during LP compared to those during H and P; 2239 +/- 702 and 1929 +/- 769, 1957 +/- 725 mg kg-1, P = 0.067, respectively. The thresholds (blood glucose level at which a sustained elevation of the counterregulatory hormones as compared to the mean at normoglycaemia level 4.0 mmol l-1, occurs) for the various hormones were very similar during LP, H, and P insulin infusions and occurred at 253.8 +/- 56.7, 256.3 +/- 55.3 and 257.5 +/- 70.0 min for adrenaline; 241.4 +/- 80.3, 260.5 +/- 82.5 and 225.0 +/- 75.9 min for noradrenaline; 307.5 +/- 65.5, 304.1 +/- 74.1 and 322.5 +/- 40.4 min for cortisol; 263.8 +/- 50.3, 255.0 +/- 63.6 and 249.6 +/- 50.9 min for growth hormone; 236.3 +/- 78.2, 200.0 +/- 73.1 and 226.3 +/- 65.5 for pancreatic polypeptide. The autonomic and neuroglycopenic symptoms were elicited at 240 and 300 min, respectively. In conclusion, our data indicate a tendency to a higher biological activity of approximately 10% for Lispro insulin. During a stepwise euglycaemic/hypoglycaemic clamp, the counterregulatory hormone responses to insulin lispro, human insulin, and porcine insulin were similar.