CONTEXT: Vigilance for threat is a key feature of generalized anxiety disorder (GAD). The amygdala and the ventrolateral prefrontal cortex constitute a neural circuit that is responsible for detection of threats. Disturbed interactions between these structures may underlie pediatric anxiety. To date, no study has selectively examined responses to briefly presented threats in GAD or in pediatric anxiety. OBJECTIVE: To investigate amygdala and ventrolateral prefrontal cortex activation during processing of briefly presented threats in pediatric GAD. DESIGN: Case-control study. SETTING: Government clinical research institute. PARTICIPANTS: Youth volunteers, 17 with GAD and 12 without a psychiatric diagnosis. MAIN OUTCOME MEASURES: We used functional magnetic resonance imaging to measure blood oxygenation level-dependent signal. During imaging, subjects performed an attention-orienting task with rapidly presented (17 milliseconds) masked emotional (angry or happy) and neutral faces. RESULTS: When viewing masked angry faces, youth with GAD relative to comparison subjects showed greater right amygdala activation that positively correlated with anxiety disorder severity. Moreover, in a functional connectivity (psychophysiological interaction) analysis, the right amygdala and the right ventrolateral prefrontal cortex showed strong negative coupling specifically to masked angry faces. This negative coupling tended to be weaker in youth with GAD than in comparison subjects. CONCLUSIONS: Youth with GAD have hyperactivation of the amygdala to briefly presented masked threats. The presence of threat-related negative connectivity between the right ventrolateral prefrontal cortex and the amygdala suggests that the prefrontal cortex modulates the amygdala response to threat. In pediatric GAD, amygdala hyperresponse occurs in the absence of a compensatory increase in modulation by the ventrolateral prefrontal cortex.