Serotonin (5-HT), found in abundance in intestinal enterochromaffin cells, has been shown to be released in response to neural, humoral, and intraluminal stimuli but the mechanisms controlling release are poorly understood. The purpose of this study was to determine whether 5-HT release induced by the muscarinic agonist, methacholine, is mediated by enteric nerves or is a direct action at the mucosal cell level. We mounted rabbit mucosal sheets containing intact submucosal neural elements, but stripped of muscularis propria and myenteric plexus, in modified Ussing chambers and measured the release of 5-HT in response to 5 X 10(-5) M methacholine added to both mucosal and serosal surface bathing solutions, in the presence and absence of selective neural blockade with 1 X 10(-6) M tetrodotoxin. 5-HT release in control tissues (no drugs) as measured by radioimmunoassay was 25.7 +/- 6 ng/cm2/30 min (mean +/- SEM). In the presence of mucosal and serosal methacholine, total 5-HT release increased significantly (P less than 0.05) to 66.7 +/- 9 ng/cm2/30 min. When tetrodotoxin alone was applied, 5-HT release significantly increased to 55.2 +/- 9 ng/cm2/30 min compared to matched control. In the presence of tetrodotoxin, methacholine increased 5-HT release to 79.3 +/- 9 ng/cm2/30 min, which was significantly greater than with tetrodotoxin alone. These results provide evidence of an inhibitory neural regulation of basal 5-HT release since release increased in the presence of neural blockade. They also suggest that a muscarinic receptor at or near the enterochromaffin cells mediates mucosal 5-HT release since 5-HT is further increased by methacholine even in the presence of neural blockade.