General anesthetics modulate autonomic nervous system function including thermoregulatory control, which resides in the preoptic area of the anterior hypothalamus. However, the mechanism by which anesthetics modulate hypothalamic function remains unknown. We hypothesized that isoflurane increases norepinephrine release in the preoptic area and in the posterior hypothalamus causing hypothermia during anesthesia. To test this hypothesis, we performed a series of in vivo and in vitro studies in rats. In vivo studies: 1) Norepinephrine release was measured by microdialysis in the preoptic area or the posterior hypothalamus (n=9 each) before, during (30 min), and after (50 min) rats were anesthetized with 2% isoflurane. 2) In five rats, blood gases and arterial pressure were measured. 3) Body temperature changes (n=6 each) were measured after prazosin (0, 0.05, 0.5 microg), norepinephrine (0, 0.1, 1.0 microg), or 0.5 microg prazosin with 1.0 microg norepinephrine injection into the preoptic area. In vitro study: Norepinephrine release was measured from anterior or posterior hypothalamic slices (n=10 each) incubated with 0, 1, 2, or 4% isoflurane in Ca2+-containing buffer or with 4% isoflurane (n=10) in Ca2+-free buffer. Data were analyzed with repeated measures or factorial ANOVA and Student-Newman-Keuls tests. P<0.05 was significant. During anesthesia, norepinephrine release in the preoptic area was increased approximately 270%, whereas the release in the posterior hypothalamus remained unchanged. During emergence, posterior hypothalamic norepinephrine release increased by approximately 250% (P<0.05). Rectal temperature changes correlated with norepinephrine release from the preoptic area. Norepinephrine in the preoptic area enhanced isoflurane-induced hypothermia, while prazosin reversed it. Norepinephrine release from anterior hypothalamic slices increased at all isoflurane concentrations, but only at the highest concentration in posterior hypothalamic slices. Under Ca2+-free conditions, 4% isoflurane increased norepinephrine from both regions. These results suggest that augmentation of norepinephrine release in the preoptic area is responsible for hypothermia during general anesthesia.