1. This study was designed to determine whether a 1 h period of mild hypoglycaemia (3.3 or 3.7 mmol/l) affected the response to an episode of moderate hypoglycaemia (2.5 mmol/l) immediately afterwards. 2. Eleven non-obese healthy men (age 26 +/- 1 years, mean +/- SEM) underwent three separate 3 h hyper-insulinaemic glucose clamps in single-blind, random order. On all three occasions, blood glucose was 4.5 mmol/l for the first hour, and on a control visit was maintained at this level for the second hour. In the other two visits, blood glucose was lowered to 3.7 or 3.3 mmol/l during the second hour. In the third hour, blood glucose was lowered to 2.5 mmol/l on all three visits. 3. In the second hour, adrenaline rose significantly (P < 0.05, analysis of variance) with a blood glucose of 3.3 and 3.7 mmol/l, as did cortisol and heart rate at 3.3 mmol/l, but glucagon, prolactin, sweating rate, symptom score and blood pressure were the same during the second hour on all three visits. 4. In the final hour at 2.5 mmol/l, there were no differences in adrenaline, noradrenaline, glucagon, prolactin, cortisol, symptom score, heart rate, blood pressure or sweating rate. 5. Thus, the overall magnitude of hormonal responses to moderate hypoglycaemia (2.5 mmol/l) are not modified by exposure to mild hypoglycaemia (3.3 or 3.7 mmol/l) for 1 h immediately beforehand.