Using high performance liquid chromatography techniques with fluorescence and electrochemical detection we found that -nicotinamide adenine dinucleotide (-NAD) is released in response to electrical field stimulation (EFS, 4-16 Hz, 0.3 ms, 15 V, 120 s) along with adenosine 5'-triphosphate (ATP) and norepinephrine (NE) in the canine isolated mesenteric arteries. The release of -NAD increases with number of pulses/stimulation frequencies. Immunohistochemistry analysis showed dense distribution of tyrosine hydroxylase-like immunoreactivity (TH-LI) and sparse distribution of TH-LI negative nerve processes, suggesting that these blood vessels are primarily under sympathetic nervous system control with some contribution of other (e.g., sensory) neurons. Exogenous NE (3 µmol/L), .-MeATP (1 µmol/L), neuropeptide Y (NPY, 0.1 µmol/L), calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP) and substance P (SP), 0.1 µmol/L, had no effect on the basal release of -NAD, suggesting that the overflow of -NAD is evoked by neither the sympathetic neurotransmitters NE, ATP, and NPY nor the neuropeptides CGRP, VIP, and SP. Botulinum neurotoxin A (BoNTA, 0.1 µmol/L) abolished the evoked release of NE, ATP, and -NAD at 4 Hz, suggesting that at low levels of neural activity release of these neurotransmitters results from SNARE/SNAP-25 protein mediated exocytosis. At 16 Hz, however, the evoked release of NE, ATP, and -NAD was reduced by BoNTA by about 90%, 60%, and 80%, respectively, suggesting that at higher levels of neural activity -NAD is likely to be released from different populations of synaptic vesicles or different populations of nerve terminals (e.g., sympathetic and sensory terminals).