The effect of a single dose (2 g/kg, body weight) of ethanol on diamine oxidase activity of the upper gastrointestinal tract was studied in fasted rats. Ethanol given by gastric intubation as a 10%, 20%, or 40% solution caused an early and transient increase in diamine oxidase activity in gastroduodenal fluid that was concentration‐dependent. The 20% ethanol solution caused, at 3 and 6 hr, a diminution of enzyme activity in the wall of the stomach and duodenum, but not in that of the jejunum. Diamine oxidase activity in the gastroduodenal tract returned to control values at 12 hr, when ethanol had disappeared from the blood. The plasma enzyme activity diminished, starting from the first hour. The same dose of ethanol administered intravenously caused diminutions in diamine oxidase activity in the stomach and duodenum similar to those observed after gastric intubation. The enzyme decrease in the stomach and duodenum was not correlated with the gastroduodenal or blood ethanol levels. Pretreatment with pyrazole, an inhibitor of ethanol metabolism, prevented the ethanol‐induced decrease in gastroduodenal enzyme activity, thus suggesting that this diminution was principally a consequence of ethanol oxidation. These results indicate that ethanol modifies in the gastroduodenal tract the activity of diamine oxidase the enzyme which regulates the physiologic activity of histamine in gastric secretion and oxidizes the toxic diamines of dietary and bacterial origin.