Actions of lubiprostone, a selective type-2 chloride channel activator, on mucosal secretion were investigated in guinea pig small intestine and colon. Flat-sheet preparations were mounted in Ussing flux chambers for recording short-circuit current (Isc) as a marker for electrogenic chloride secretion. Lubiprostone, applied to the small intestinal mucosa in 8 concentrations ranging from 1 to 3000nM, evoked increases in Isc in concentration-dependent manner with an EC₅₀ of 42.5nM. Lubiprostone, applied to the mucosa of the colon in 8 concentrations ranging from 1 to 3000nM evoked increases in Isc in concentration-dependent manner with an EC₅₀ of 31.7nM. Blockade of enteric nerves by tetrodotoxin did not influence stimulation of Isc by lubiprostone. Antagonists acting at prostaglandin PGE₂, EP_1-3 or EP₄ receptors did not suppress stimulation of Isc by lubiprostone, but suppressed or abolished PGE₂-evoked responses. Substitution of gluconate for chloride abolished all responses to lubiprostone. The selective CFTR channel blocker, CFTR(inh)-172, did not suppress lubiprostone-evoked Isc. The broadly-acting blocker, glibenclamide, suppressed (P<0.001) lubiprostone-evoked Isc. Lubiprostone, in the presence of tetrodotoxin, enhanced carbachol-evoked Isc. The cholinergic component, but not the putative VIP component, of neural responses to electrical field stimulation was enhanced by lubiprostone. Application of either of the prostaglandins, E₂, F₂ or I₂, evoked depolarization of the resting membrane potential in enteric neurons. Unlike the prostaglandins, lubiprostone did not alter the electrical behavior of enteric neurons. Exposure to the histamine H₂ receptor agonists increased basal Isc followed by persistent cyclical increases in Isc. Lubiprostone increased the peak amplitude of the dimaprit-evoked cycles.