The present study was done to determine the influence of dietary fat on the effect of ethanol on pancreatic macromolecular content and secretion. Weight-matched groups of Sprague-Dawley rats were divided into controls fed Rodent-Blox ad libitum; American Institute of Nutrition-76 (AIN-76) diet containing 12% calories as fat with 36% of carbohydrate calories replaced with 5% (weight/volume) concentration of ethanol fed ad libitum pair fed with animals given isocaloric amounts of AIN-76 diet for three to six months. Compared with Rodent-Blox fed controls, tissue content of trypsinogen, chymotrypsinogen, amylase, and lipase; specific activity and concentration of trypsinogen, chymotrypsinogen; and concentration of amylase were decreased at six months in AIN-76 fed controls. These changes did not result from diminished food intake, but were due to adaptation to the liquid diet. Animals fed AIN-76 diet plus ethanol did not show significant difference in the total content, specific activity, concentration, and secretion of digestive enzymes compared with those animals pair fed isocaloric amounts of AIN-76 diet. Activation of trypsinogen by exogenous trypsin was lower in rats fed AIN-76 diet and a similar change was observed in animals fed AIN-76 diet with ethanol for six months. These findings are in contrast to increased secretion of proteases and decreased trypsin inhibitor observed previously in animals fed ethanol in a diet containing "high" fat. These data indicate that ethanol effect on the pancreas is modified by dietary intake of fat and/or carbohydrates.