The activities of 2- and 4-hydroxylation enzymes of estradiol 17-sulfate in various rat tissues were measured by determining 2-hydroxyestradiol 17-sulfate and 4-hydroxyestradiol 17-sulfate by high performance liquid chromatography equipped with an electrochemical detector. Incubations were carried out in the presence of a nicotinamide adenine dinucleotide phosphate (NADPH)-generating system using microsomal fractions of each tissue. The livers of male and female rats, followed by the kidneys, had the highest 2-hydroxylase activities, while other tissues, including the brain, heart, lung, testis, ovary and uterus, possessed similar levels of activity. With regard to 4-hydroxylase, again the liver, followed by the ovary, had the highest activity. There was no significant difference in the activities of other tissues investigated. In addition, two catechol estrogens, 6 beta-, 7 beta- and 16 alpha-hydroxylated metabolites of estradiol 17-sulfate, were formed when male heart and lung and female liver and kidney were used. Male kidney was found to have a weak 6 beta-hydroxylase activity, also. These results show that various rat tissues, including the liver, have 2- and 4-hydroxylase activities for estradiol 17-sulfate and that some of the tissues have additional metabolic abilities such as hydroxylation at 6 beta-, 7 beta- or 16 alpha-positions of the substrate.