Co-activation of alpha2 adrenergic receptors (AR) and opioid receptors (OR) produces antinociceptive synergy. The antinociceptive synergy between intrathecally administered alpha2 AR agonists and OR agonists has been well documented. The mechanism of how such agonists synergize remains unclear. One possibility is that the synergism between two receptors is mediated in a single subcellular compartment. Purification of spinal nerve terminals enables discrete single compartmental interrogation of protein-protein interactions. The dependent measures observable include 1) neurotransmitter/modulator uptake mechanisms into pre-synaptic neurons, 2) depolarization-mediated release of neurotransmitter or neuromodulator, and 3) inhibition of depolarization-mediated release of neurotransmitter or neuromodulator by application of agonists. We used this preparation to evaluate the transport kinetics and release dynamics of a decarboxylated form of arginine, agmatine, which is a potential endogenous ligand for the imidazoline and NMDA receptors as well as nitric oxide synthase enzyme. These studies used L-arginine as a negative control and the metabolically stable analog of glutamate, D-aspartate, as a positive control for depolarization-dependent release. We also evaluated the ability of the delta opioid receptor-selective agonist deltorphin II to inhibit D-aspartate release (as the primary experimental variable) when delivered alone or in combination with alpha2 AR receptor agonists. Agmatine, L-arginine, and D-aspartate all accumulated in spinal cord synaptosomes in a temperature-dependent manner. KCl and capsaicin-evoked depolarization caused Ca2+-dependent release of agmatine and D-aspartate (but not L-arginine). Deltorphin II inhibited the release of potassium-evoked release of D-aspartate, These data demonstrate that stimulation of synpatosomes of spinal cord origin by KCl results in measurable release of neuromodulator/neurotransmitter. Such release is inhibited by activation of the delta opioid receptor, which is consistent with its suggested localization to presynaptic nerve terminals.