There is a lack of drugs currently available that directly target epithelial fluid and electrolyte transport to treat secretory diarrhoea. We have previously shown that ursodeoxycholic acid (UDCA), widely used in cholestatic liver disease, exerts antisecretory actions on colonic epithelial cells in vitro However, in vivo, UDCA does not persist in the colon but undergoes bacterial metabolism to lithocholic acid (LCA) which, in contrast, enhances secretion. We hypothesised that metabolically stable UDCA analogs should have antisecretory actions in vivo AIMS: To examine molecular mechanisms underlying the antisecretory response to UDCA and test whether dehydroxylation-resistant analog, 6-methylUDCA (6MUDCA) exerts antisecretory effects in vivo. METHODS: Cl- secretion was measured as changes in short circuit current across voltage-clamped T84 cell monolayers and muscle-stripped mouse colon in Ussing chambers. Confocal microscopy and surface biotinylation assessed abundance/surface expression of transport proteins. Data are presented as mean ± SEM, and statistically analysed by Students t-test/ANOVA. RESULTS: In T84 cells UDCA (500 µM) attenuated secretory responses to Ca2+- and cAMP-dependent agonists, carbachol (CCh; 100µM) and forskolin (FSK; 10µM) to 3.4 ± 0.7% (n = 9; p < 0.001) and 43.0 ± 13% (n = 6; p < 0.05), respectively. Effects were concentration-dependent over the range 50 µM – 1 mM. UDCA inhibited CCh-induced responses within 1 min, while >15 mins was required to inhibit FSK-induced responses, suggesting involvement of multiple signaling pathways. UDCA was not cytotoxic as determined by lactate dehydrogenase release and transepithelial resistance measurements. The antisecretory effect of UDCA in T84 cells was duplicated by analog 6MUDCA. To determine molecular mechanisms involved, we examined effects of UDCA on components of the Cl- secretory pathway. UDCA inhibited Na+/K+-ATPase pump activity to 16.2±4.0% (n = 4; p < 0.001) of controls but did not alter cellular abundance or surface expression of the protein. UDCA also inhibited basolateral K+ channel current to 13.7±4%(n = 7; p<0.001) but did not alter apical Cl- channel current. Intraperitoneal injection of UDCA (100 mg/kg; 4 hrs) significantly potentiated secretory responses in ex vivo colonic tissue from mice, consistent with the hypothesis that it is metabolized to LCA. However, similar treatment with 6MUDCA reduced CCh-induced responses to 52.5 ± 14.7% of controls (n = 5; p < 0.001). CONCLUSIONS: These data indicate that stable UDCA analogs exert antisecretory actions in vivo, likely through inhibition of multiple components of the Cl- secretory pathway and thus may represent a new class of anti-diarrhoeal drug directly targetting epithelial transport.