This report presents novel results on the effects of serotonin (5-HT) on longitudinal muscle contractions in the rabbit ileum and the interactions of serotonin with some neuronal elements of the myenteric plexus. We showed previously that serotonin-triggered contractions involved two mechanisms in the rabbit ileum: neuronal excitation (via 5-HT2 receptors in the neurons) and direct muscular stimulation (via 5-HT4 receptors in the muscle). Here, we focus on the neuronal 5-HT2 receptor pathway and report further pharmacological and immunocytochemical data clarifying the details of the mechanisms. We observed that antagonists for neurokinin (NK1 and NK2) receptors partially blocked the serotonin response, but NK3 receptor antagonists had no effect. Pretreatment by atropine (ATR) eliminated the NK1 receptor antagonist resistant contractions. In contrast, the NK1 antagonist did not depress the ATR-resistant contraction when ATR was added first. 5-HT2 receptor agonist-induced contractions were partially suppressed by ATR, hexamethonium, and NK1 or NK2 receptor antagonists. In conclusion, serotonin acting through 5-HT2 receptors could stimulate interneurons and excitatory motor neurons. Immunocytochemical staining revealed an extensive tachykinin-immunoreactive (IR) network in the myenteric plexus. Approximately 52% of all myenteric neurons were labeled. 5-HT-IR fibers could be detected around both choline acetyltransferase- and tachykinin-IR cells, suggesting functional relationships between them. Consistent with our pharmacological observations, we found that immunopositive nerve elements for 5-HT2A receptor and double-labeled immunostaining revealed a remarkable overlap between tachykinin-IR neurons and 5-HT2A-IR elements. Anat Rec, 2009. © 2009 Wiley-Liss, Inc.