Intrathecal agmatine (decarboxylated arginine) moderates induction of neuropathic pain, spinal cord injury, and opioid tolerance in rodents. An endogenous central nervous system molecule and N-methyl-D-aspartate receptor antagonist/nitric oxide synthase inhibitor, agmatine may be a neuromodulator. We evaluated depolarization-induced release of agmatine from purified spinal nerve terminals (synaptosomes). Agmatine immunoreactivity was observed colocalized or closely apposed to some synaptophysin- and/or synaptotagmin-labeled structures. A temperature- and concentration-dependent uptake of [3H]-agmatine into synaptosomes was observed, consistent with an uptake mechanism. Potassium-induced depolarization resulted in release of [3H]-agmatine from the synaptosomes in a Ca2+-dependent manner, consistent with a neuromodulatory function. These results agree with previous reports of agmatine uptake into synaptosomes of the brain and extend those results to include stimulated release and a spinal site of activity.