The effect of ethanol on polyamine acetylation was studied in rat liver. Animals were fed on nutritionally complete liquid diets with 36% or 12% of total calories supplied as ethanol or isocaloric carbohydrates for 4 months. The diet with 36% calories as ethanol significantly increased the activity of cytosolic spermidine/spermine N1-acetyltransferase, the rate-limiting enzyme in polyamine interconversion. Such a stimulation did not appear in rats under the 12% ethanol regimen. The stimulation of spermidine/spermine N1-acetyltransferase was associated with increases in putrescine and spermidine concentrations, an appearance of N1-acetylspermidine, and a decrease in spermine. These results suggest that chronic ethanol intake stimulates the acetylation of polyamines and their interconversion in rat liver.