Catecholestrogens are biologically active metabolites of estrogen which are synthesized by estrogen-2-hydroxylase in the liver, brain, and other organs. Although catecholestrogens are inactivated by enzymatic O-methylation, demethylation of the 2-methoxyestrogens to reform catecholestrogens also occurs. A sensitive and specific radioenzymatic assay for rat liver 2-methoxyestrogen demethylase has been developed which makes it possible to characterize its substrate requirements and to study the influence of various hormones on catecholestrogen formation. 2-Methoxyestrogen demethylase activity is localized in rat liver microsomes. The apparent Km for 2-methoxyestrone (2MeOE1) is 12 μM and that for 2-methoxyestradiol (2MeOE2) is 3 μM. The two most prevalent 2-methoxyestrogens, 2MeOE1 and 2 MeOE2, appear to be demethylated by different enzymes. The enzymes have absolute requirements for NADPH, and their activities are inhibited by CO and SKF-525A, indicating that they are cytochrome P450 dependent. 2MeOE2 demethylation but not 2MeOE1 demethylation exhibits substrate inhibition. 2MeOE1 demethylase activity in the female rat liver is only one third that in the male rat liver, but sexual dimorphism was not found in 2MeOE2 demethylation. Thyroidectomy and estradiol treatment of the male rat resulted in diminished 2MeOE1, but not 2MeOE2, demethylation.