In mammalian intestine, a number of secretagogues have been shown to work through either cyclic nucleotide or calcium mediated pathways to elicit ion secretion. Because excessive intestinal electrolyte and fluid secretion is central to the pathogenesis of a variety of diarrheal disorders, understanding of these processes is essential to the development of future clinical treatments. In the current study, the effects of serotonin (5HT), histamine, and carbachol on intestinal ion transport were examined in in vitro preparations of rabbit ileum. All three agonists induced a rapid and transient increase short-circuit current (delta Isc) across ileal mucosa. Inhibition of the delta Isc response of all three agents in chloride-free solution or in the presence of bumetanide confirmed that chloride is the main electrolyte involved in electrogenic ion secretion. Pretreatment of tissue with tetrodotoxin or atropine did not effect secretagogue-mediated electrolyte secretion. While tachyphylaxis of delta Isc response was shown to develop after repeated exposure of a secretagogue to tissue, delta Isc responses after sequential addition of different agonists indicate that cross-tachyphylaxis between agents did not occur. Serotonin, histamine, and carbachol have previously been reported to mediate electrolyte secretion through calcium-dependent pathways. To access the role of extracellular calcium in regulating ion secretion, the effect of verapamil on each agent was tested; verapamil decreased 5HT-induced delta Isc by 65.2% and histamine response by 33.5%, but had no effect on carbachol-elicited chloride secretion. An additive secretory effect was found upon simultaneous exposure of 5HT and carbachol to the system; no other combination of agents produced a significant additive effect. Findings from this study support previous work which has suggested that multiple calcium pathways may be involved in mediating chloride secretion in mammalian intestine.