To study the effect of acute stress on ACTH secretion and synthesis in rat pituitary and hypothalamus, ACTH content and POMC mRNA levels (measured by use of Northern blot analysis) in these tissues as well as the levels of ACTH in plasma and those of CRF in the hypothalamus were determined after insulin-induced hypoglycemia. Plasma ACTH levels increased at 30 and 60 min. ACTH levels in the anterior pituitary lobe (AP) decreased at 30 min, and then returned to control levels at 60 min. No change was seen in the intermediate-posterior pituitary (IP) or the hypothalamus after insulin injection. CRF levels decreased at 30 and 60 min, then returned to control levels at 90 min in the medial basal hypothalamus, including the median eminence. Hybridization with a cDNA probe revealed a single size class of POMC mRNA in AP, IP, and hypothalamus, and the size of POMC mRNA in these tissues did not change during the experimental period. POMC mRNA levels in AP increased at 60 min and reached a peak at 120 min, but those in IP and hypothalamus did not change. These results suggest that 1) insulin-induced hypoglycemia stimulates both secretion and synthesis of ACTH (at least by increasing POMC mRNA levels) in the AP, and 2) the levels of ACTH and POMC mRNA in the IP and hypothalamus are not affected by insulin-induced hypoglycemia.