Abstract Although it is well known that there are sex differences in stress-induced activation of the hypothalamic–pituitary–adrenal axis, it is not known if there are also gender-related differences in stress-induced neural activity. In this study, restraint and formalin injections into a forelimb were used as stressors and 2-[ 14C]deoxyglucose (2DG) autoradiography was used to evaluate regional brain glucose metabolism, an index of neural activity. Analysis of blood samples collected during the 2DG procedure confirmed that stress elevates plasma glucose levels signficantly more in females than in males. Moreover, females show higher brain glucose utilization in all regions examined, including sex hormone-responsive regions such as the medial amygdala, medial preoptic nucleus, ventromedial nucleus, and arcuate nucleus, as well as the CA1 layer and dentate region of the hippocampus, the posterior parietal (sensorimotor) cortex, medial and lateral habenula, and splenium of the corpus callosum. The sex differences are apparent regardless of whether animals were injected with saline or formalin. Interestingly, the medial preoptic area, which shows robust neuroanatomical sex differences, demonstrates greater activation in response to formalin than to saline only in females. In some regions of both males and females, glucose utilization was higher on the side of the brain contralateral to the saline or formalin injection site. These findings suggest that there are widespread, gender-related differences in neuronal as well as endocrine activation in response to highly stressful conditions.