Water-handling epithelia are sensitive to the osmotic environment. In this study, the effects of a hypo-osmotic challenge on carbachol (CCh)-induced fluid secretion was investigated using an ex vivo submandibular gland perfusion technique and intracellular pH and Ca²⁺ measurements. The osmolality of the perfusion solution was altered to examine the response of the gland to a hypotonic challenge. The flow rate was increased by 34% with a 30% hypotonic solution (225 mOsm), although the Ca²⁺ response was unchanged. Lowering the external Cl^- by 50% abolished this increase in the 30% hypotonic solution. Furthermore, bumetanide, an inhibitor of the Na⁺-K⁺-2Cl^- co-transporter, completely inhibited the fluid secretion increase caused by the 30% hypotonic solution, and both the total amount of fluid and the flow rate were identical to those of the isotonic solution. This finding was confirmed by measuring Na⁺-K⁺-2Cl^- co-transporter, bumetanide-dependent NH₄⁺ transport; Na⁺-K⁺-2Cl^-- transport was up-regulated more than 40% by a 30% hypotonic challenge. Therefore, the increase in CCh-induced fluid secretion in response to hypotonic conditions can be attributed to a large extent to the specific activation of the Na⁺-K⁺-2Cl^- co-transporter.