Intestinal bacterial metabolism of the catechol estrogens, 2-hydroxyestrone (2-OHE1), 2-methoxyestrone (2-MeOE1) and 2-hydroxyestradiol (2-OHE2), was studied by incubation of the steroid with mixed fecal flora. 3-Methoxyestrone (3-MeOE1) was included in order to evaluate whether demethylation occurs also with regard to noncatecholic estrogens. The purification of the samples and separation into fractions was carried out by ion exchange chromatography and the metabolites formed were identified by combined gas chromatography-mass spectrometry (GC-MS). Mixed human fecal flora was able to interconvert 2-OHE1 and 2-OHE2 both in aerobic and anaerobic conditions. Demethylation occurred both for 2-MeOE1 and 3-MeOE1.