These experiments were designed to determine whether fasting and feeding were associated with differing rates of protein synthesis in the rat pancreas. It has been established that feeding, acetylcholine, or cholecystokinin-pancreozymin administration was associated with enhanced rates of digestive enzyme secretion; however, the literature is unclear as to effects of such stimulation on enzyme synthesis. Rats fed ad lib. or fasted 24, 48, or 72 hr were used for this study. Pancreases were removed and incubated in tissue culture medium with l-phenylalanine-(14)C, and incorporation into TCA-insoluble material as well as purified amylase was measured. Compared with fed controls, fasting 24, 48, and 72 hr was associated with 29%, 39%, and 35% decreases in incorporation of l-phenylalanine-(14)C into protein. Decreases of similar magnitudes were apparent whether the data were expressed in terms of protein or DNA. Pancreatic amylase isolated from rats fasted 48 hr contained 57% fewer counts of l-phenylalanine-(14)C than amylase isolated from fed rats. Moreover, rats fasted for 24 hr and given bethanechol chloride incorporated greater amounts of l-phenylalanine-(14)C into protein than fasted controls. Studies were performed to exclude changes in pool size of precursor (l-phenylalanine-(14)C) or product (amylase) in accounting for decreases associated with fasting. These studies demonstrate that fasting was associated with decreased rates of pancreatic amylase and protein synthesis in rats.