The capacity of the intestine to secrete fluid is dependent on the basolateral Na+-K+-2Cl- co-transporter (NKCC1). Given that cAMP and calcium signals promote sustained and transient episodes of fluid secretion, respectively, this study investigated the differential regulation of functional NKCC1 membrane expression in the native human colonic epithelium. Tissue sections and colonic crypts were obtained from sigmoid rectal biopsy tissue samples. Cellular location of NKCC1, Na/K-ATPase, M3AChR and lysosomes was examined by immunolabelling techniques. NKCC1 activity (i.e. bumetanide-sensitive NH4+ uptake), intracellular calcium and cell volume were assessed by BCECF, Fura2 and DIC/calcein imaging. Unstimulated NKCC1 was expressed on basolateral membranes and exhibited a topological expression gradient, predominant at the crypt-base. Cholinergic calcium signals initiated at the crypt-base and spread along the crypt-axis. In response, NKCC1 underwent a calcium-dependent 4-hour cycle of recruitment to basolateral membranes, activation, internalisation, degradation and re-expression. Internalisation was prevented by the EGFR kinase inhibitor tyrphostin-AG1478 and re-expression was prohibited by the protein synthesis inhibitor cylcoheximide; the lysosome inhibitor chloroquine promoted accumulation of NKCC1 vesicles. NKCC1 internalisation and re-expression was accompanied by secretory volume decrease and bumetanide-sensitive regulatory volume increase, respectively. In contrast, Forskolin (i.e. cAMP elevation) -stimulated NKCC1 activity was sustained, and membrane expression and cell volume remained constant. Co-stimulation with forskolin and acetylcholine promoted dramatic recruitment of NKCC1 to basolateral membranes and prolonged the cycle of co-transporter activation, internalisation and re-expression. In conclusion, persistent NKCC1 activation by cAMP is constrained by a calcium-dependent cycle of co-transporter internalisation, degradation and re-expression; a novel mechanism to limit intestinal fluid loss.