Hepatic microsomal carboxylesterase (EC 3.1.1.1) from rat liver microsomes showed a different capacity for the hydrolysis of various substrates. In hypophysectomized male rats, hepatic carboxylesterase activities showed substrate-dependent changes, i.e., an decrease in malathion and p-nitrophenylacetate hydrolases and increase in isocarboxazid hydrolase. When human growth hormone is administered to hypophysectomized male rats, carboxylesterase activities were decreased. Conversely, subcutaneous injection of ovine prolactin to hypophysectomized rats, carboxylesterase activities showed substrate-dependent changes. On the other hand, in hypophysectomized male rats, the amount of carboxylesterase isozymes showed different changes, i.e., an increase in the amount of RL2 contents and decrease in RL1 contents, and the amount of RH1 was not changed. These results suggested that hepatic microsomal carboxylesterases are regulated, at least in part, by some pituitary hormones, and that these hormones have selective effects on the different isozymes of carboxylesterase.