Selective serotonin reuptake inhibitors are first-line treatment for most anxiety disorders, but their mechanism of anxiolytic action has not been clarified. Selective serotonin reuptake inhibitors are anxiolytic in conditioned fear stress (re-exposure to an environment paired previously with inescapable electric footshocks). To clarify the brain regions where selective serotonin reuptake inhibitors act, we examined the effect of microinjection of the selective serotonin reuptake inhibitor, citalopram, into the amygdala, medial prefrontal cortex and mediodorsal nucleus of the thalamus on freezing behavior, an index of fear, induced by conditioned fear stress. Bilateral injection of citalopram into the amygdala before testing reduced freezing significantly, while bilateral injection into the medial prefrontal cortex or mediodorsal nucleus of the thalamus did not. These results suggest that the anxiolytic effect of a selective serotonin reuptake inhibitor in conditioned fear is mediated by its effect in the amygdala, and support the hypothesis of serotonin function in anxiety by which facilitation of serotonin neurotransmission decreases anxiety.