The effects of ethanol on pancreatic digestive and lysosomal enzymes may be relevant to the pathogenesis of alcoholic pancreatitis since pancreatic enzymes are thought to play an important role in the development of pancreatic injury. Previous studies, using the Lieber-DeCarli pair-feeding model of ethanol administration, have demonstrated that ethanol significantly increases the content and gene expression of pancreatic enzymes. However, these findings have been questioned because, in the Lieber-DeCarli model, ethanol-fed rats have a lower carbohydrate intake than their pair-fed controls, making it difficult to ascribe any observed changes to ethanol alone. This study was designed to distinguish between the effects of ethanol and those of reduced dietary carbohydrate on pancreatic enzymes, using a quartet-feeding model of ethanol administration. Rats were fed liquid diets containing low (11%) and high (47%) amounts of carbohydrate, with and without ethanol, for four weeks. The effects of ethanol on pancreatic content and messenger RNA levels for digestive enzymes (trypsinogen, chymotrypsinogen and lipase) and a lysosomal enzyme (cathepsin B) were assessed. Ethanol feeding resulted in a significant increase in glandular content with a corresponding increase in mRNA levels for all four enzymes studied. By contrast, a reduction in dietary carbohydrate intake alone did not alter pancreatic content or gene expression for the above enzymes. These results indicate that (i) ethanol significantly increases the capacity of the acinar cells to synthesise digestive enzymes and the lysosomal enzyme cathepsin B, and (ii) these changes are due to ethanol itself and are not due to variations in dietary carbohydrate intake.