Ouabain (10(-3) M) caused a 95.8% reduction in the volume of saliva secreted during a 60-min period by the isolated, perfused submandibular gland of the rat exposed to acetylcholine (10(-6) M) and modified salivary cation (Na and K) concentrations but not salivary Cl concentrations. Furosemide (10(-3) M) caused a 74.9% reduction in saliva volume and significantly reduced salivary Cl concentrations but did not modify salivary Na or K. Ethacrynic acid (10(-4) M) resulted in a 58.6% reduction in saliva volume, increased salivary Na and Cl concentrations, and reduced salivary K+ concentrations at low rates of flow. The results suggest that an ouabain-sensitive Na+-K+-ATPase and a furosemide-sensitive NaCl cotransport system contribute to acetylcholine-induced fluid secretion in the rat submandibular gland. The Na+-K+-ATPase probably provides the energy or driving force for the NaCl cotransport system by maintaining a Na+ gradient in the salivary cells. The lesser effect of ethacrynic acid on saliva volumes may result from a quantitatively smaller action on the same NaCl cotransport affected by furosemide. An ouabain-sensitive pump present in salivary ducts regulates transductal transport of Na and K.