1. This study investigates whether the cholinergic neurones, innervating the human proximal stomach, can be modulated by nitric oxide (NO) or vasoactive intestinal polypeptide (VIP), or via presynaptic muscarinic, alpha(2)- or 5-hydroxytryptamine(4) (5-HT(4)-) receptors. 2. Circular muscle strips, without mucosa, were incubated with [(3)H]-choline to incorporate [(3)H]-acetylcholine into the cholinergic transmitter stores. The basal and electrically-induced release of tritium and [(3)H]-acetylcholine were analysed in a medium containing guanethidine (4 x 10(-6) M), hemicholinium-3 (10(-5) M), physostigmine (10(-5) M) and atropine (10(-6) M). Tissues were stimulated twice for 2 min (S(1) and S(2): 40 V, 1 ms, 4 Hz) and drugs were added before S(2). 3. The NO synthase inhibitor L-N(G)-nitroarginine methyl ester (3 x 10(-4) M) and the NO donor sodium nitroprusside (10(-5) M), as well as VIP (10(-7) M) did not influence the basal release nor the electrically-evoked release. 4. The alpha(2)-adrenoceptor agonist UK-14,304 (10(-5) M) significantly inhibited the electrically-evoked release of [(3)H]-acetylcholine, and this was prevented by the alpha(2)-adrenoceptor antagonist rauwolscine (2 x 10(-6) M). 5. The 5-HT(4)-receptor agonist prucalopride (3 x 10(-7) M) significantly enhanced the electrically-evoked release of [(3)H]-acetylcholine, and the 5-HT(4)-receptor antagonist SB204070 (10(-9) M) prevented this. 6. When atropine (10(-6) M) was omitted from the medium and added before the second stimulation, it significantly increased the release of [(3)H]-acetylcholine. 7. These results suggest that the release of acetylcholine from the cholinergic neurones, innervating the circular muscle in the human proximal stomach, can be inhibited via presynaptic muscarinic auto-receptors and alpha(2)-adrenoceptors, and stimulated via presynaptic 5-HT(4)-receptors. No evidence for modulation by NO or VIP was obtained.