Bile acids are important regulators of physiological processes in the intestine, ranging from inflammation to ion transport. While studies have shown that deoxycholic acid and chenodeoxycholic acid cause rapid prosecretory actions on colonic epithelia, the monohydroxy bile acid, lithocholic acid (LCA), appears to be devoid of prosecretory activity. However, LCA is always present in the colonic lumen and its levels can be dramatically altered. Thus, it is important to investigate the consequences of prolonged exposure to LCA or its metabolite, 7-keto LCA, on colonic epithelial barrier and transport function. Cl- secretion, the primary driving force for intestinal fluid secretion, was measured as changes in short circuit current (Isc) across voltage-clamped monolayers of T84 cells. Transepithelial electrical resistance (Rte) was measured using an EVOM2 apparatus. Acid phosphatase (AP) activity was employed as a measure of toxicity. Results are expressed as mean ± SEM. Statistical analyses were made by one way ANOVA and Tukey multiple comparisons test. p values ≤ 0.05 were considered to be significant. Bilateral treatment of T84 monolayers with LCA for 24 hrs resulted in a concentration-dependent reduction in Rte, being abolished by LCA (500μM), whereas physiologically-relevant concentrations of LCA (≤10μM) were without significant effect. AP activity was abolished by LCA (≥100μM), whereas LCA (10μM) significantly reduced AP activity (p≤0.01). The Ca2+-dependent agonist, carbachol (CCh), stimulated Cl- secretory responses that were reduced by LCA (10μM)-pretreatment to 50.4% compared to controls (p≤0.001), whereas the cAMP-dependent agonist, forskolin (FSK), stimulated secretory responses that were reduced by non-toxic concentrations of LCA (1μM and 100nM) to 78.0 and 82.9% compared to controls, respectively (p≤0.001). Treatment of T84 monolayers with the LCA metabolite, 7-keto LCA, for 24 hrs also resulted in a concentration-dependent reduction in Rte. However, in contrast to LCA, 7-keto LCA at high concentrations (500μM) only partially reduced Rte(p≤0.05), whereas 7-keto LCA (10μM) was without effect. AP activity was also reduced in a concentration-dependent manner, but was significantly attenuated only at a concentration of 5 mM (p≤0.05). CCh-stimulated secretory responses were reduced by 7-keto LCA (500μM) to 30.5% (p≤0.001) compared to controls, while FSK-stimulated responses were reduced by 7-keto LCA (100 μM) to 72.5% compared to controls (p≤0.05). These studies provide new insights into the roles that bile acids play in regulation of colonic epithelial barrier and transport function. Further studies to elucidate the role of LCA and 7-keto LCA in regulating colonic epithelial barrier and transport function in health and disease are important and may help in the development of new therapeutic approaches for intestinal transport disorders.