Based on findings in rodents, we sought to test the hypothesis that purinergic modulation of synaptic transmission occurs in the human intestine. Time-series analysis of intraneuronal free Ca²⁺ levels in SMP from Roux-en-Y specimens was done using Zeiss LSM laser-scanning confocal Fluo-4/AM-Ca²⁺ imaging. A 3sec fiber tract stimulation (FTS) was used to elicit a synaptic Ca²⁺ response. Short-circuit current (I_sc=chloride secretion) was recorded in mucosa-SMP in flux chambers. A distension reflex or EFS was used to study I_sc responses. Ca²⁺ imaging was done in 1,222 neurons responding to high K⁺ depolarization from 61 surgical cases. FTS evoked synaptic Ca²⁺ responses in 62% of recorded neurons. FTS caused frequency ()-dependent Ca²⁺ responses (0.1-100Hz). FTS Ca²⁺ responses were inhibited by -conotoxin (70%), hexamethonium (50%), TTX , high Mg²⁺ /low Ca²⁺ ( 100%) or capsaicin (25%). A P2Y1R antagonist MRS 2179 or PLC inhibitor U73122 blocked FTS responses (75-90%). P2Y1R-ir occurred in 39% VIP-positive neurons. The selective ADOA3R agonist 2-Cl-IBMECA caused concentration- and -dependent inhibition of FTS Ca²⁺ responses (IC₅₀= 8.5x10^-8 M). The ADOA3R antagonist MRS 1220 augmented such Ca²⁺ responses; 2-Cl-IBMECA competed with MRS 1220. Knock-down of ADOA1R with FSCPX did not prevent 2-Cl-IBMECA effects. MRS 1220 caused 31% augmentation of TTX-sensitive distension I_sc-responses. The SMP from Roux-en-Y patients is a suitable model to study synaptic transmission in huENS. The P2Y1 Gq-PLC/IP3 Ca²⁺ signaling pathway, N-Ca²⁺ channels, nicotinic receptors and extrinsic nerves contribute to neurotransmission in huENS. Inhibitory ADOA3 receptors modulate nucleotide and cholinergic transmission in the huENS.