Many anxiety disorders, as well as major depressive disorder (MDD), are at least twice as prevalent in women as in men, but the neurobiological basis of this discrepancy has not been well studied. MDD is often precipitated by exposure to uncontrollable stress, and is frequently characterized by abnormal or disrupted prefrontal cortex (PFC) function. In animals, exposure to stress has been shown to cause PFC dysfunction, but sex differences in this effect have not been investigated. The present study tested male and female rats on a PFC-dependent working memory task after administration of FG7142, a benzodiazepine inverse agonist that activates stress systems in the brain. Female rats were impaired by lower doses than males during proestrus (high estrogen), but not during estrus (low estrogen). Similarly, ovariectomized females showed increased stress sensitivity only after estrogen replacement. These results suggest that estrogen amplifies the stress response in PFC, which may increase susceptibility to stress-related disorders.