In nerve–smooth muscle preparations β-nicotinamide adenine dinucleotide (β-NAD) has emerged as a novel extracellular substance with putative neurotransmitter and neuromodulator functions. β-NAD is released, along with noradrenaline and adenosine 5′-triphosphate (ATP), upon firing of action potentials in blood vessels, urinary bladder and large intestine. At present it is unclear whether noradrenaline, ATP and β-NAD are stored in and released from common populations of synaptic vesicles. The answer is unattainable in complex systems such as nerve–smooth muscle preparations. Adrenal chromaffin cells are thus used here as a single-cell model to examine mechanisms of concomitant neurosecretion. Using high-performance liquid chromatography techniques with electrochemical and fluorescence detection we simultaneously evaluated secretion of dopamine (DA), ATP, adenosine 5′-diphosphate, adenosine 5′-monophosphate, adenosine, β-NAD and its immediate metabolites ADP-ribose and cyclic ADP-ribose in superfused nerve growth factor-differentiated rat pheochromocytoma PC12 cells. β-NAD, DA and ATP were released constitutively and upon stimulation with high-K+ solution or nicotine. Botulinum neurotoxin A tended to increase the spontaneous secretion of all substances and abolished the high-K+-evoked release of β-NAD and DA but not of ATP. Subcellular fractionation by continuous glycerol and sucrose gradients along with immunoblot analysis of the vesicular marker proteins synaptophysin and secretogranin II revealed that β-NAD, ATP and DA are stored in both small synaptic-like vesicles and large dense-core-like vesicles. However, the three substances appear to have different preferential sites of release upon membrane depolarization including sites associated with SNAP-25 and sites not associated with SNAP-25.