Whole body exposure to high doses of ionizing radiation is associated with small intestinal and colonic dysfunction, the etiology of which remains unknown. In this study, we investigated the role of both neural and nonneural 5-hydroxytryptamine (5-HT)-mediated pathways in radiation-induced attenuation and recovery of colonic secretory function. Rats were exposed to whole body 10-Gy gamma irradiation, and distal colonic tissues were studied in Ussing chambers 1, 3, and 7 days after exposure. Tissue responses to exogenously added 5-HT (nonneural pathway) and electrical field stimulation (EFS; neural pathway) were performed, and 5-HT receptor subtypes implicated in both responses were determined using three different 5-HT receptor antagonists: methysergide (5-HT_2/1C), granisetron (5-HT₃), and SDZ-205,557 (5-HT₄). Maximal responses to exogenously added 5-HT were decreased at 1 and 3 days and returned to control values at 7 days. Responses to exogenous 5-HT were insensitive to both 5-HT_2/1C and 5-HT₃ antagonists and to TTX but were totally inhibited by SDZ-205,557 in both control and irradiated tissues. Responses to EFS were decreased 1 and 3 days after exposure and returned to control values at 7 days. In control tissues and 1 and 3 days after exposure, EFS responses were insensitive to both 5-HT_2/1C and 5-HT₄ antagonists but reduced by granisetron in control (51%) and at 1 (64%) and 3 days (58%) after exposure. Granisetron was more effective at 7 days (73% inhibition), which was concomitant with the appearance of a 5-HT₄ antagonist-sensitive pathway (40% inhibition). In conclusion, neural and nonneural 5-HT-mediated pathways involve 5-HT₃ and 5-HT₄ receptors, respectively, in control as well as in irradiated tissues 1 and 3 days after exposure. Conversely, the recovery of colonic transport is associated with additional 5-HT₃-mediated pathways, probably in combination with 5-HT₄ receptors.