The role of Na+ in fluid secretion by the isolated rabbit pancreas was investigated. The fluid secretion rate is reduced upon replacement of Na+ in the bathing medium by Li+, K+ or choline. The inhibition depends on the nature of the substituting cation, and is largest with choline. Upon replacement, the substituent cation appears in the secreted fluid, and the Na+ concentration in the secreted fluid is decreased in a mirror-like fashion. When Na+ is replaced by Li+ or choline, the secretory Na+ concentration is decreased, although less than in the bathing medium, and the K+ concentration is increased. When Na+ is replaced by K+, the Na+ and the K+ concentration in the secreted fluid are approximately equal to their bathing medium concentrations. In the Li+ and choline medium, stimulation of the pancreas by carbachol or CCK-8 increases the fluid secretion rate. In addition, it increases the Li+ or choline concentration, and decreases the Na+ and K+ concentrations in the secreted fluid. In normal and K+ medium, stimulation causes only a slight increase in fluid secretion rate, with no change in the secretory Na+ concentration. In normal medium, stimulation leads to a decrease in the secretory K+ concentration. The effects of replacing Na+ appear to be the result of a direct inhibition of the active HCO3- transport underlying secretion, and an indirect inhibition related to the permeability of the pancreas for the various cations. The stimulants are likely to act by increasing the permeability of the tight junctions.