An assay of cholesteryl ester hydrolase activity of microsomes from guinea pig adrenal indicated maximal activity at pH at pH 4.0 and 5.5. When ethanol was added to the microsomes, these enzymes showed differences in response with increasing ethanol concentration. Rats administered 15% ethanol as drink for 4--5 months showed a small (20%) increase in cholesteryl ester hydrolase (pH 4.0) activity in adrenal as compared to controls. However, a 50% decrease in cholesteryl ester hydrolase activity resulted when rats were administered ethanol by intragastric tubing technique (15 days) as compared to controls treated similarly with saline. The decrease in cholesteryl ester hydrolase activity was correlated with an increase in cholesteryl ester level in serum. In a different study, rats intubated with an acute dose of ethanol showed an increase in adrenal cholesteryl ester hydrolase activity as early as 1 hr after administration. This effect seemed to last for about 24 hr before activity returned to control level. It is concluded that different modes of ethanol administration to rats may elicit a change in cholesteryl ester hydrolase activity in the adrenal. An increase in cholesteryl ester hydrolase activity may be related to an increase in steroidogenic activity and activation of adrenocortical function.