Recent studies from our laboratory (Ponnappa et al. (1988) Biochim. Biophys. Acta 966, 390–402), indicate that in the pancreas of rats fed ethanol chronically, an overnight withdrawal of ethanol stimulated the rate of protein synthesis in vivo, whereas, during continuous ethanol ingestion, the rate of protein synthesis was the same as in the control group which did not receive ethanol. However, a stimulation of protein synthesis was also observed when isolated acini were prepared from the pancreas of continuously ethanol-fed rats. In the present studies, preparations of pancreatic lobules were used to further characterize the stimulatory effect observed in the ethanol-fed group. The rate of protein synthesis was studied in vitro by determining the rate of incorporation of [ 3H]leucine into proteins. Similar to in vivo observations, chronic ethanol feeding did not alter the rate of protein synthesis, but an overnight withdrawal of ethanol stimulated the rate of protein synthesis by 84%. The stimulation of protein synthesis refelcted a general enhancement in the rate of synthesis of most of the digestive enzymes ranging from 60 to 110%. The maximal stimulation of protein synthesis occured within 24 h of ethanol withdrawal and the rates rapidly decreased to control levels within 3 days. During the overnight ethanol withdrawal there was also 30–40% decrease in the activities of most of the pancreatic digestive enzymes. This observation indicates that ethanol withdrawal also initiated the secretion and/or degradation of pancreatic digestive enzymes in vivo. The observation that the enhanced rate of protein synthesis can be observed in isolated acini but not in vivo or in lobular preparations from the continuously ethanol-fed rats indicates that the pancreas contains factors which supress this stimulatory effect of ethanol intake. The stimoulation of protein synthesis, brought about either by ethanol withdrawal or by collagenase digestion of the tissue, may reflect the removal of such factors.