In an attempt to clarify the role of the brain serotonergic system in the psychopathology of anxiety, we examined the effect of a psychological stress, conditioned fear stress, on extracellular serotonin (5-hydroxytryptamine, 5-HT) concentrations in the rat medial prefrontal cortex using the method of in vivo microdialysis, while simultaneously observing conditioned fear stress-induced freezing behavior, an index of anxiety. Conditioned fear stress increased extracellular 5-HT levels in the medial prefrontal cortex, and this 5-HT level increase was followed by a resolution of the freezing behavior. A dose of 10 mg/kg of a selective 5-HT reuptake inhibitor, citalopram, administered 60 min before exposure to conditioned fear stress increased extracellular 5-HT concentrations immediately and potently, reducing freezing behavior. These findings strongly suggest that facilitation of brain 5-HT neurotransmission decreases anxiety, which is in agreement with the clinical reports that selective 5-HT reuptake inhibitors are effective in the treatment of anxiety disorders.