Chronic ethanol consumption causes decreased hepatic protein degradation, resulting in protein accumulation within hepatocytes. In this investigation, we sought to determine whether chronic ethanol feeding alters the degradative capacity and protease activities of isolated hepatic lysosomes. Male Sprague-Dawley-derived rats were fed a liquid diet containing either ethanol (36% of calories) or isocaloric maltose-dextrin for 1-5 wk. Hepatic lysosomes were isolated by differential centrifugation and purified through Percoll gradients. Lysosomes obtained from livers of ethanol-fed rats degraded both endogenous protein substrates and the exogenously added radioactive substrate, 125I-RNase A, 26-42% more slowly than lysosomes from pair fed controls. The ethanol-elicited reduction in proteolytic capacity appeared to result in part, from a deficiency of the lysosomal cathepsins B, L, and H. Compared with controls, the specific activities of these enzymes were 31-45% lower in lysosomes from ethanol-fed rats. Immunoblot analyses also revealed that the intralysosomal as well as the intracellular content of cathepsin B was significantly lower in ethanol-fed rats. In contrast, ethanol consumption did not affect the cellular quantity of cathepsin L but lowered its amount in isolated lysosomes. Our findings suggest that chronic ethanol consumption causes a deficiency in lysosomal cathepsins by altering their biosynthesis and/or their trafficking into lysosomes.