OBJECTIVE: The hypothalamus is the central brain region responsible for sensing and integrating responses to changes in circulating glucose. The aim of this study was to determine the time sequence relationship between hypothalamic activation and the initiation of the counterregulatory hormonal response to small decrements in systemic glucose. RESEARCH DESIGN AND METHODS: Nine nondiabetic volunteers underwent two hyperinsulinemic clamp sessions in which pulsed arterial spin labeling was used to measure regional cerebral blood flow (CBF) at euglycemia ( approximately 95 mg/dl) on one occasion and as glucose levels were declining to a nadir of approximately 50 mg/dl on another occasion. Plasma glucose and counterregulatory hormones were measured during both study sessions. RESULTS: CBF to the hypothalamus significantly increased when glucose levels decreased to 77.2 +/- 2 mg/dl compared with the euglycemic control session when glucose levels were 95.7 +/- 3 mg/dl (P = 0.0009). Hypothalamic perfusion was significantly increased before there was a significant elevation in counterregulatory hormones. CONCLUSIONS: Our data suggest that the hypothalamus is exquisitely sensitive to small decrements in systemic glucose levels in healthy, nondiabetic subjects and that hypothalamic blood flow, and presumably neuronal activity, precedes the rise in counterregulatory hormones seen during hypoglycemia.