Biochemical information about receptors for adrenergic and opioid neurotransmission in submucosal plexus (SMP) is unavailable. We have purified a fraction P2 enriched in synaptosomes and neuronal membranes (high [3H]saxitoxin binding and high vasoactive intestinal polypeptide immunoreactivity, low activity of 5'-nucleotidase) from the canine small intestine SMP. The synaptosomal fraction (fraction P2) also contained a high density of opioid diprenorphine binding sites of high affinity. [3H]rauwolscine binding was enriched both in fraction P2 and in a microsomal fraction. Competition experiments using several adrenergic and opioid receptor ligands revealed that opioid receptors were approximately 64% mu-, 24% delta-, and 12% kappa-subtypes and that adrenoceptors on fraction P2 were alpha 2-subtype but that there was a heterogeneous population of alpha 2-adrenoceptors. These studies show that a fraction enriched in synaptosomes and neural membranes from the canine intestine SMP contains opioid as well as alpha 2-adrenoceptors, that all three subtypes of opioid receptors seem to be present with mu-receptors predominant, and that subtypes of alpha 2-adrenoceptors appear to be present.