Most newborn mammals, including humans, are susceptible to neonatal hypoglycemia shortly after birth, especially if fasted. Glycogenolysis provides short term benefit, but gluconeogenesis is required for glucose homeostasis at this stage of development. A functional hypothalamic-pituitary-adrenocortical (HPA) axis is essential for glucose homeostasis in adults, but little is known of the importance of this endocrine axis in this regard during the neonatal period. The neonatal rat is an excellent model for study of both glucose metabolism and the HPA, and shares many similarities with other mammals. For the first 2 weeks of postnatal life, the rat HPA appears to be relatively unresponsive to stress, however, which would seem maladaptive during the critical first 24 h. The present study examined the ability of day-old rats to respond to hypoglycemia with activation of the HPA system. Each of three doses of insulin resulted in hypoglycemia (glucose, less than 40 mg/dl), with the greatest decrease in glucose 120 min after an insulin dose of 2.5 U/kg BW. At this dose, plasma corticosterone was increased to levels comparable to those observed in adults. The steroid response was associated with a small but significant increase in ACTH, and both endocrine responses were delayed compared to those in adults. There was no difference in adrenal response time to ACTH in incubations of adrenal cells isolated from day-old or adult rats, however. The stimulatory effect of insulin was progressively diminished in older animals (2-6 days postnatal). Insulin had no direct effect on adrenal steroidogenesis either alone or in combination with ACTH when tested on dispersed cells in vitro. We conclude that 1) the adrenal gland is very sensitive to the stress of hypoglycemia for 1 day after birth, but rapidly declines in activity over the first 6 days of life; 2) the response time of the HPA axis to hypoglycemia stress is delayed in day-old rats, and this delay occurs above the level of the adrenal; and 3) there may be factors in addition to ACTH that promote steroidogenesis on the first postnatal day, or there may be bioactive or immunoreactive differences in the ACTH molecule during this time.