The distribution of orexin A-immunoreactive neurons and orexin type I receptors in the CNS suggests important roles in regulating the hypothalamo-pituitary gonadal (HPG) axis and sexual behaviors. We examined orexin A interactions in the HPG axis in vivo and in vitro. Orexin A stimulated LH-releasing hormone (LHRH) release in hypothalamic explants harvested from male rats (+133%) and from females at proestrus (+233%), with no effect at estrus or metestrus. Orexin A dose dependently inhibited LHRH-stimulated LH release in dispersed pituitaries from proestrous females only. A selective NPY1-receptor antagonist abolished in vitro release of LHRH by orexin A. Hyperestrogenization in female rats reduced orexin A content in hypothalamus (−28%), midbrain (−26%), medulla (−40%), thalamus (−36%), olfactory tubercles (−25%), and cortex (−35%), brain regions that are important in HPG control and sex-cycle specific behaviors. Orexin A content was lower in hypothalamus (−20%) and higher in midbrain (+40%), medulla (+31%), and thalamus (+33%) at late proestrus vs. other cycle stages. Orexin A release after administration of 56 mm KCl was significantly greater in hypothalamic explants harvested on the morning of proestrus than at estrus or metestrus, and orexin A release was stimulated by estradiol (E2) in explants from males. These results reveal important interactions for orexin A in the HPG axis.