The alpha2 adrenergic receptor (α2-AR) antagonist yohimbine is a widely used tool for the study of anxiogenesis and stress-induced drug-seeking behavior. We previously demonstrated that yohimbine paradoxically depresses excitatory transmission in the bed nucleus of the stria terminalis (BNST), a region critical to the integration of stress and reward pathways, and produces an impairment of extinction of cocaine-conditioned place preference (cocaine-CPP) independent of α2-AR signaling. Recent studies show yohimbine-induced drug-seeking behavior is attenuated by orexin receptor 1 (OX1 R) antagonists. Moreover, yohimbine-induced cocaine-seeking behavior is BNST-dependent. Here, we investigated yohimbine-orexin interactions. Our results demonstrate yohimbine-induced depression of excitatory transmission in the BNST is unaffected by alpha1-AR and corticotropin-releasing factor receptor-1 (CRFR1) antagonists, but is (1) blocked by OxR antagonists and (2) absent in brain slices from orexin knockout mice. Although the actions of yohimbine were not mimicked by the norepinephrine transporter blocker reboxetine, they were by exogenously applied orexin A. We find that, as with yohimbine, orexin A depression of excitatory transmission in BNST is OX 1 R-dependent. Finally, we find these ex vivo effects are paralleled in vivo, as yohimbine-induced impairment of cocaine-CPP extinction is blocked by a systemically administered OX1 R antagonist. These data highlight a new mechanism for orexin on excitatory anxiety circuits and demonstrate that some of the actions of yohimbine may be directly dependent upon orexin signaling and independent of norepinephrine and CRF in the BNST. © 2012 American College of Neuropsychopharmacology. All rights reserved.