We have examined the distribution of L-ascorbic acid in rat parotid acinar cells by analysis of whole tissue, subcellular fractions, and parotid salivary secretion. Acinar cell secretion granules contain reduced ascorbate at a calculated concentration of 3.5 mM. A distinct extragranular pool, comprising as much as 80% of total cellular ascorbate, also exists in these cells. Parotid secretion, collected from the cannulated duct after inducing secretion from acinar cells by isoproterenol administration, contains millimolar reduced ascorbate. Quantitatively, the level measured in parotid secretion, relative to the secreted enzyme alpha-amylase, is nearly identical to that measured in isolated granules, suggesting a common release by exocytosis. Although ascorbate has been extensively studied as a content component of adrenal chromaffin granules and has recently been implicated in secretion granules of other neural and endocrine tissues, its detection in secretion granules of exocrine cells is novel. Thus, ascorbate-dependent processes that occur in exocrine secretion granules, or that may be general to all types of secretion granules, are worthy of consideration.