The incubation of PE2 ((6,7-3H)-labelled 3-propyl ether of estra-1,3,5(10)triene-3,17β-diol (estradiol)) with various subcellular fractions of rat liver indicated that the hepatic metabolism of this compound occurs mainly in the microsomal fraction.In addition to the formation of 3-propyl ethers of estra-1,3,5(10)triene-3-ol-17-one (estrone) and estra-1,3,5(10-triene-3,16α,17β-triol (estriol) directly deriving from PE2, the microsomal proteins carried out the deetherification of the propyl ether group leading to phenolic steroids; among them, estradiol, estrone, and estriol were characterized. Protein-bound and water-soluble metabolites were found; the effects of glutathione and of the incubation conditions were in agreement with the thioconjugation of these derivatives.The microsomal metabolism of PE2, and specially the deetherification reaction, required the presence of oxygen and of NADPH as cofactor, the optimum pH ranging from 7.4 to 8. The participation of cytochrome P450 in these metabolic pathways was shown by a partially inhibited catabolism with carbon monoxide and by a more active metabolism in males than in females and when animals were pretreated with phenobarbital.These results allowed us to conclude that the hepatic deetherification of PE2 is carried out by a microsomal oxidative system which is very similar to the system involved in the demethylation of methyl ethers of estrogens.