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The aim of this study was to identify the receptor type(s) by which 5-hydroxytryptamine applied to the intestinal mucosa excites the terminals of myenteric AH neurons. The AH neurons have been identified as the intrinsic primary afferent (sensory) neurons in guinea-pig small intestine and 5-hydroxytryptamine has been identified as a possible intermediate in the sensory transduction process. Intracellular recordings were taken from AH neurons located within 1 mm of intact mucosa to which 5-hydroxytryptamine was applied. Trains of action potentials and/or slow depolarizing responses were recorded in AH neurons in response to mucosal application of 5-hydroxytryptamine (10 or 20 μM) or the 5-hydroxytryptamine-3 receptor agonist, 2-methyl-5-hydroxytryptamine (1 or 3 mM), and to electrical stimulation of the mucosa. The 5-hydroxytryptamine-2 receptor agonist, α-methyl-5-hydroxytryptamine (100 μM), and the 5-hydroxytryptamine-1,2,4 receptor agonist, 5-methoxytryptamine (10 μM), did not elicit such responses. The 5-hydroxytryptamine-3 receptor-selective antagonist, granisetron (typically 1 μM), and the 5-hydroxytryptamine-3,4 receptor antagonist, tropisetron (typically 1 μM), each reduced or abolished the responses to 5-hydroxytryptamine, while the selective 5-hydroxytryptamine-4 receptor antagonist, SB 204070 (1 μM), did not. It is concluded that application of 5-hydroxytryptamine to the mucosa activates a 5-hydroxytryptamine-3 receptor that triggers action potential generation in the mucosal nerve terminals of myenteric AH neurons.