A single injection of ethanol was shown to produce a relatively rapid increase in the in vitro activity of aniline hydroxylase and in microsomal ethanol oxidation. Stimulation of the microsomal ethanol oxidating system (MEOS) was dependent on the ethanol dose and time after dosing. Hepatic MEOS activities were significantly increased 2, 3 and 4 hr after a single 4.0 g/kg i.p. dose of ethanol and 4 hr after administration of 2.0 and 3.25 g/kg doses. Renal MEOS activity was also significantly increased 4 hr after a 4.0 g/kg dose of ethanol. Hepatic MEOS and aniline hydroxylase activities per gram of liver were significantly increased 2 hr after a 4.0 g/kg dose of ethanol, whereas microsomal cytochromes P-450 and b5, NADPH cytochrome c reductase, protein and NADPH-stimulated and ethanol/NADPH-stimulated lipid peroxidation were unchanged. Cycloheximide pretreatment did not block ethanol-induced stimulation of aniline hydroxylase or MEOS activity. Finally, an acute injection of ethanol (4.0 g/kg) produced significantly higher MEOS activities per gram of liver (121% of control) in mice chronically ingesting ethanol than in mice treated with saline. This investigation shows that MEOS activity can respond rapidly to a dose of ethanol and that synthesis of new protein is not responsible for this stimulation of activity.