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The role of vitamin D in parotid gland function was investigated by measuring the composition and rate of production of parotid saliva in response to pilocarpine injection in vitamin-D-deficient and replete rats in vivo. Rats fed a vitamin-D-free diet from weaning (G1) were studied after 8 weeks of diet at which time they had a decreased rate of growth, were hyperparathyroid and hypocalcaemic but still had detectable serum 1,25-dihydroxycholecalciferol (1,25(OH)2D3). Rats which were the offspring of vitamin-D-deficient mothers and which were maintained on a vitamin-D-free diet from weaning (G2) had a decreased rate of growth from birth, were hypocalcaemic and hyperparathyroid and at no time had any detectable serum 1,25(OH)2D3. In response to pilocarpine, the volume of parotid saliva produced by G1 animals was no different from the controls (G1 animals receiving supplemental vitamin D) whereas that produced by G2 animals was reduced more than 65%. The total amount of amylase secreted was unchanged in either group of experimental animals so that the concentration of amylase in the parotid saliva from G2 animals was increased. The concentration of calcium in parotid saliva changed in parallel with the changes in serum calcium in G1 and G2 animals. It is concluded that the primary source of parotid saliva calcium is the extracellular fluid and not zymogen granules and the transepithelial transport of this calcium is independent of vitamin D; the secretion of electrolytes and water, which in the parotid gland require extracellular calcium, is dependent on vitamin D. It is proposed that the vitamin is necessary for the synthesis of a protein(s) which is essential for the utilization of extracellular calcium in this secretion process.