The muscarinic acetylcholine receptor (mAChR) molecular subtype, m2, has been postulated to be the presynaptic cholinergic autoreceptor in many brain regions. However, due to a lack of subtype-specific pharmacological agents, conclusive evidence for m2 as an autoreceptor remains elusive. The development of subtype-specific antibodies has enabled extensive characterization of the synaptic localization of the m2 subtype. Specifically, double-labeling immunocytochemistry with m2 antibodies and antibodies to the vesicular acetylcholine transporter (VAChT), a novel specific marker of cholinergic terminals, in the striatum has allowed the first direct anatomical evidence of m2 localization in cholinergic terminals. Additionally, other anatomical studies in striatum and the septohippocampal pathway have revealed that this subtype is also expressed presynaptically in non-cholinergic terminals, and is postsynaptically expressed in both cholinergic and non-cholinergic neurons. The implications of these data for understanding the functional roles of this subtype are discussed.