The effect of a single feeding of ethanol on hepatic ornithine decarboxylase (ODC) activity in rats was investigated. Ethanol (7.5 g/kg body weight) was tube‐fed to overnight‐fasted rats as a 50% (v/v) solution in water 1, 2, 3, 4, 8, 12, or 24 hr before sacrifice. The levels of ODC activity in the livers were assayed in vitro by measuring the release of14C02from 0L‐1‐14C‐ornithine. Hepatic ODC activities were significantly stimulated by ethanol (7.5 g/kg body weight) beginning at 1 hr and reaching a peak at 4 hr (more than a 16‐fold increase over zero time controls). Single feedings of varying doses of ethanol (2.5, 5.0, or 7.5 g/kg body weight) to overnight‐fasted rats 3 hr before sacrifice also exhibited significant increases (3 to 13‐fold) in the hepatic ODC activities. In vitro14C‐leucir>e incorporation into protein using hepatic microsomes of ethanol‐treated rats was decreased in comparison with that of controls. The ethanoMnduced stimulation of hepatic ODC activity was not abolished by pretreatment with pyrazoie, an inhibitor of ethanol metabolism. However, the stimulation of hepatic ODC activity by ethanol was suppressed by actinomycin D or cycloheximide, indicating that the enhancement is attributable to the synthesis of new enzyme protein. Furthermore, abolition of the stimulation of hepatic ODC activity due to ethanol by prior adrenalectomy suggests that the induced increase is probably mediated through stimulation of adrenal hormones. These studies demonstrate that a single dose of ethanol per os can significantly enhance in the rat the activity of hepatic ODC, a key enzyyme in the biosynthesis of polyamines, and that the effect is indirect, v