The effect of glucagon on the activity of alcohol dehydrogenase in rat hepatocyte culture was determined. Glucagon concentrations of 0.1 nM enhanced, whereas concentrations greater than 1 nM decreased, alcohol dehydrogenase. These effects became apparent after exposure of the cultures to glucagon for 4 or more days. The presence of corticosterone (1 microM) prevented the enhancing effect of 0.1 nM glucagon on alcohol dehydrogenase activity. The changes in alcohol dehydrogenase caused by glucagon were associated with parallel changes in the rate of ethanol elimination. Alcohol dehydrogenase appears to be rate-limiting for ethanol oxidation, as uncoupling of oxidative phosphorylation did not modify the rate of ethanol elimination. These studies suggest a physiologic role of glucagon in enhancing liver alcohol dehydrogenase activity, whereas higher pharmacologic concentrations of glucagon have an opposite, depressant effect.