The submucosal plexus of the enteric nervous system of the gut contains neurons that regulate water and electrolyte secretion, which are essential for maintaining body fluid balance. The submucosal plexus of the guinea pig small intestine contains 2 major classes of secretomotor neurons: vasoactive intestinal peptide (VIP) and cholinergic neurons which release VIP or acetylcholine, respectively to stimulate epithelial ion transport. Purines acting on P2Y1 receptors (P2Y1R) are implicated in mediating neurogenic intestinal secretion (1). Current literature proposes that P2Y1Rs selectively excite VIP neurons in guinea pig small intestine (1, 2) particularly since P2Y1Rs mediate intermediate and slow excitatory postsynaptic potentials in VIP secretomotor neurons (2-4). However, potential effects on cholinergic secretomotor neurons are not excluded and the specific role of P2Y1R remains unclear. We examined the secretory role of P2Y1R in guinea pig jejunum. Short-circuit current (Isc), indicative of chloride secretion, was measured across mucosa-submucosa preparations in Ussing chambers. Agonists and antagonists were applied to the serosal half-chamber. Values are presented as mean ± SEM and compared by one-way ANOVA. P<0.05 was considered significant. VIP and cholinergic/choline acetyltransferase (ChAT)-containing submucosal neurons were identified by immunofluorescence. 2MeSADP (50 nM; P2Y1 agonist) evoked an increase in Isc (n = 14) that was near abolished by tetrodotoxin (1 μM, Na+ channel blocker; n=5). The 2MeSADP-evoked response was inhibited 55 ± 9% by PPADS (30 μM, P2Y antagonist; n = 6) and 47 ± 9% by the VPAC1R (VIP receptor subtype 1) antagonist, PG97-269 (1 μM; n = 11). Hyoscine (10 μM; muscarinic antagonist) reduced the 2MeSADP response by 42 ± 6% (n = 6). Hyoscine and PG97-269 combined reduced the 2MeSADP response by 84 ± 7% (n = 6). A small proportion of submucosal neurons coexpressed VIP and ChAT (n = 4 animals). Our data indicate that activation of P2Y1R on submucosal neurons causes VIP- and acetylcholine-release. This is the first demonstration that P2Y1R-mediated secretion involves cholinergic secretomotor neurons. Contrary to the well-established segregation of VIP and cholinergic submucosal neurons in guinea pig ileum, we observed some overlap of VIP and ChAT immunoreactivity in jejunum. This may account for the cholinergic component of the 2MeSADP response. However, it is also possible that 2MeSADP activates cholinergic secretomotor neurons indirectly, via VIP release within the submucosal plexus stimulating VPAC1R on cholinergic secretomotor neurons (5). We conclude that secretion evoked by P2Y1R activation involves cholinergic and VIP neurons.