Delta opioid receptors (DORs) are associated with ischemic preconditioning and vagal transmission in the SA node and atria. Although functional studies suggested DORs are prejunctional on parasympathetic nerve terminals, their precise location remains unconfirmed. DORs were co-localized in tissue slices and synaptosomes from the canine right atrium and SA node along with cholinergic and adrenergic markers, vesicular acetylcholine transporter (VAChT) and tyrosine hydroxylase (TH). Synapsin I immunofluorescence verified the neural character of tissue structures and isolated synaptosomes. Acetylcholine and norepinephrine measurements suggested the presence of both cholinergic and adrenergic synaptosomes. Fluorescent analysis of VAChT and TH signals indicated that more than 80% of the synapsin positive synaptosomes were of cholinergic origin and less than 8% were adrenergic. DORs co-localized 75-85% with synapsin in both tissue slices from atria and SA node. The co-localization was equally strong (85%) for nodal synaptosomes but less so for atrial synaptosomes (57%). Co-localization between DOR and VAChT was 75-85% regardless of the source. Overlap between DOR and TH was uniformly low ranging from 8-17%. Western blots with synaptosomal extracts confirmed two DOR positive bands at molecular weights corresponding to those reported for DOR monomers and dimers. The abundance of DOR was greater in nodal synaptosomes than in atrial synaptosomes; largely attributable to a greater abundance of monomers in the SA node. The abundant nodal and atrial DORs predominantly associated with cholinergic nerve terminals supports the hypothesis that prejunctional DORs regulate vagal transmission locally within the heart.