Recent evidence supports an emerging role of β-nicotinamide adenine dinucleotide (β-NAD+) as a novel neurotransmitter and neuromodulator in the peripheral nervous system –β-NAD+ is released in nerve-smooth muscle preparations and adrenal chromaffin cells in a manner characteristic of a neurotransmitter. It is currently unclear whether this holds true for the CNS. Using a small-chamber superfusion assay and high-sensitivity high-pressure liquid chromatography techniques, we demonstrate that high-K+ stimulation of rat forebrain synaptosomes evokes overflow of β-NAD+, adenosine 5′-triphosphate, and their metabolites adenosine 5′-diphosphate (ADP), adenosine 5′-monophosphate, adenosine, ADP-ribose (ADPR) and cyclic ADPR. The high-K+-evoked overflow of β-NAD+ is attenuated by cleavage of SNAP-25 with botulinum neurotoxin A, by inhibition of N-type voltage-dependent Ca2+ channels with ω-conotoxin GVIA, and by inhibition of the proton gradient of synaptic vesicles with bafilomycin A1, suggesting that β-NAD+ is likely released via vesicle exocytosis. Western analysis demonstrates that CD38, a multifunctional protein that metabolizes β-NAD+, is present on synaptosomal membranes and in the cytosol. Intact synaptosomes degrade β-NAD+. 1,N 6-etheno-NAD, a fluorescent analog of β-NAD+, is taken by synaptosomes and this uptake is attenuated by authentic β-NAD+, but not by the connexin 43 inhibitor Gap 27. In cortical neurons local applications of β-NAD+ cause rapid Ca2+ transients, likely due to influx of extracellular Ca2+. Therefore, rat brain synaptosomes can actively release, degrade and uptake β-NAD+, and β-NAD+ can stimulate postsynaptic neurons, all criteria needed for a substance to be considered a candidate neurotransmitter in the brain.