Cell line models of colonic electrolyte transport have been extensively used despite lacking some characteristics of the native tissue. While native colonic crypts absorb or secrete NaCl, immortalized cell lines only retain the secretory phenotype. In the present study we have characterized functionally and molecularly, vectorial fluid and electrolyte transport in the morphologically differentiated human colonic cell line LIM1863. LIM1863 cells form morphologically differentiated organoids resembling native human colonic crypts, which secrete fluid and electrolytes across the apical membrane into a centrally-located lumen. Net fluid secretion was evaluated by means of morphometric measurement of lumens formed in LIM organoids in response to known secretagogues. Pharmacological profiling of the channels and transporters involved in fluid and electrolyte transport showed that net fluid transport requires Cl- uptake across the basolateral membrane through a Na+-K+-2Cl- cotransporter (NKCC1) and its subsequent exit across an apical cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. Similar to the native colon, net Cl- secretion in the LIM1863 cell line is activated by cAMP-mediated agonists. Carbachol, a Ca2+-mediated agonist, does not induce net Cl- secretion but modulates the cAMP-activated response. Expression of chloride channels (CFTR and the Ca2+-dependent Cl- channel, ClCa1), potassium channels (KCNN4 and KCNQ1), epithelial Na+ channel (ENaC) a, b and g subunits and ion transporters (NKCC1; anion exchanger, AE2; Na+/H+ exchangers, NHE1-3) was detected by RT-PCR and western blot in the case of ENaC. Based on this evidence we propose that LIM1863 cells provide a unique model for studying CFTR-dependent Cl- secretion in a morphologically differentiated human colonic crypt cell line that also expresses ENaC.