The actions of serotonin and substance P have been examined with use of an isolated, vascularly perfused rabbit ileal preparation. The vascular perfusate was composed of a modified Krebs' buffer solution that contained washed human red blood cells (hematocrit, 15% to 20%) and 3% albumin, with no added hormones or peptides. Ileal blood flow was held constant at 49.3 +/- 3.1 ml/min per 100 gm wet weight of intestine. Net intestinal water and electrolyte fluxes were calculated by means of an isosmotic buffer that contained carbon-14 polyethylene glycol as a nonabsorbable volume marker. Viability of this isolated perfused ileal preparation was confirmed on the basis of light microscopy, oxygen consumption, and transmucosal potential difference measurements. Control experiments, without exogenous hormone infusion, resulted in a stable preparation with a basal secretory state. Intra-arterial serotonin at 2.5 micrograms/min (n = 10) significantly stimulated secretion of H2O, Na+, and Cl- (p less than 0.01). Intra-arterial substance P at 2.5 x 10(-1) micrograms/min (n = 7) significantly increased the secretion of H2O, Na+, and Cl- (p less than 0.02). The dose of serotonin was designed to yield serotonin levels that resembled those found circulating in patients with carcinoid syndrome. These data indicate that serotonin and substance P are potent secretagogues in a mammalian system, independent of their effect on mesenteric blood flow and in the absence of extra-intestinal hormonal and neural influences.