The regulation of transepithelial fluid transport is of the utmost interest because excessive fluid secretion is associated with numerous intestinal conditions, including enteric infections and inflammatory bowel disease. Under normal circumstances, constitutive fluid absorption predominates over fluid secretion and serves to limit fluid loss from the body. Intestinal fluid secretion is driven by active transcellular Cl− transport which provides the osmotic impetus for passive fluid flow across the polarized epithelium; Cl− efflux across the apical membrane is mediated by the cystic fibrosis transmembrane regulator (CFTR) Cl− channel, and basolateral Cl− uptake is mediated by a Na+/K+/2Cl− co-transporter, NKCC1. Classically, CFTR has been considered the primary site of regulation. However, basolateral transport pathways can independently regulate transcellular Cl− transport and thus the capacity for fluid secretion/diarrhoea. NKCC1 is emerging as a central integrator of cellular signals that determine the secretory status of the intestinal epithelium. Co-transporter activity is regulated by changes in cell volume and intracellular Cl− which modulate its phosphorylation status. Secretagogues also regulate NKCC1 activity via modulation of basolateral membrane expression. Investigation of transporter expression, trafficking and activity in the native intestinal epithelium has been hampered by the short-term viability of epithelial tissue ex vivo. We have developed a 3D culture model of the native human colonic epithelium that maintains its tissue morphology and cellular polarity in culture. Fluorescence imaging techniques have demonstrated the dynamic and differential regulation of NKCC1 by calcium and cAMP second messengers. In response to cholinergic calcium signals, NKCC1 enters a cycle of recruitment to basolateral membranes, followed by transporter activation, internalization, degradation and re-expression. The routes of NKCC1 trafficking and the consequences for intestinal fluid transport will also be discussed.