Recent evidence suggests that the aromatization of testosterone to estrogen is important for the organizing effects of neonatal testosterone on neuroendocrine responses to acute challenges. However, the extent to which neonatal inhibition of aromatase alters the stress-induced activation of neural pathways has not been examined. Here we assessed central patterns of c-fos mRNA induced by 30 min of restraint in 65-d-old adult male rats that were implanted with sc capsules of the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD), introduced within 12 h of birth and removed on d 21 of weaning. Neonatal ATD decreased the expression of arginine vasopressin within extrahypothalamic regions in adults, confirming reduced estrogen exposure during development. As adults, ATD-treated animals showed higher corticosterone responses at 30 min of restraint exposure compared with control animals as well as higher c-fos expression levels in the paraventricular nucleus of the hypothalamus. ATD treatment also increased stress-induced c-fos within several limbic regions of the forebrain, in addition to areas involved in somatosensory processing. Based on these results, we propose that the conversion of testosterone to estrogen during the neonatal period exerts marked, system-wide effects to organize adult neuroendocrine responses to homeostatic threat.