AbstractExtracellular nucleotides modulate synaptic transmission and neuronal communication by activating purinergic 2 (P2) (nucleotide) receptors. Here, we assessed changes in the regulation by nucleotides and their receptors of an important physiological response – release and uptake of catecholamines – that accompanies sympathoadrenal neuronal differentiation. Nerve growth factor (NGF)‐promoted differentiation of pheochromocytoma 12 (PC12) cells enhanced the ability of the non‐hydrolyzable ATP analog, ATPγS, to stimulate catecholamine (norepinephrine, NE) release and this enhancement occurred without a significant alteration in NE uptake. In addition to ATPγS, 2‐MeSATP and αβMeATP, P2X receptor‐selective agonists, caused greater NE release from NGF‐differentiated than from undifferentiated PC12 cells. NGF‐differentiated PC12 cells had altered mRNA expression of several P2Y and P2X receptors but protein expression was only increased for P2X, in particular P2X1‐4,receptors and P2X, but not P2Y, receptor inhibitors blunted the NGF‐promoted enhancement in nucleotide‐regulated catecholamine release. Surprisingly, siRNA directed against P2X2, the receptor with the highest expression, failed to alter NE release by ATPγS. These findings indicate that sympathetic neuronal differentiation by NGF increases both the expression of P2X receptor sub‐types and their regulation of catecholamine release. NGF‐promoted increased expression of P2X receptors thus appears to be a physiologically important response that characterizes sympathetic neuronal differentiation.