Stress is a major driving force in reinstatement of drug-seeking behavior. The bed nucleus of the stria terminalis (BNST) has been identified as a key brain region in this behavior, and receives a dense input of the stress-neurotransmitter norepinephrine through the ventral noradrenergic bundle. Activation of α2-adrenergic receptors (α2-ARs) in the BNST blocks stress-induced reinstatement of drug-seeking, indicating a potentially important role for these receptors. Currently, it is unclear how α2-AR agonists elicit this behavioral action, or through which α2-AR subtype. Activation of α2-ARs decreases glutamatergic transmission in the BNST, an effect which is nearly absent in the α2A-AR knockout mouse. Here, we take advantage of a knock-in mouse in which a hemagglutinin-tagged α2A-AR was inserted into the endogenous locus, along with the α2A-AR selective agonist guanfacine, to further study the role of the α2A-AR subtype in modulation of neurotransmission in the BNST. Using immunohistochemistry, we find that α2A-ARs are highly expressed in the BNST, and that this expression is more similar in distribution to the vesicular glutamate transporters than to either norepinephrine transporter or tyrosine hydroxylase positive terminals. Using whole cell patch-clamp recordings, we show that guanfacine causes a depression of evoked excitatory and, to a more limited extent, inhibitory fast synaptic transmission. In total, these data support a prominent heterosynaptic role for α2A-ARs in modulating fast synaptic transmission in the BNST. © 2009 IBRO.