The neurotransmitters serotonin and corticotrophin-releasing factor are thought to play an important role in fear and anxiety behaviors. This study aimed to determine the relationship between corticotrophin-releasing factor-evoked changes in serotonin levels within discrete regions of the limbic system and the expression of fear behavior in rats. The effects of corticotrophin-releasing factor administration to the serotonin cell body regions of the dorsal raphe nucleus on fear behavior, behavioral activity, and extracellular serotonin levels were assessed in freely moving rats with microdialysis probes implanted into the central nucleus of the amygdala and the medial prefrontal cortex. Infusion of corticotrophin-releasing factor (0.5 microg) into the dorsal raphe rapidly induced freezing behavior, which was positively correlated with an immediate increase in serotonin release in the central nucleus of the amygdala. In contrast, cessation of freezing behavior correlated with a delayed and prolonged increase in serotonin release within the medial prefrontal cortex. Our findings suggest that corticotrophin-releasing factor-induced freezing behavior is associated with regionally and temporally distinct serotonergic responses in the limbic system that may reflect differing roles for these regions in the expression of fear/anxiety behavior.