Abstract A survey was conducted to define the sites of 17β-hydroxysteroid oxidoreductase activity in organs and tissues of male and female BALB c mice, as well as the favored direction of the oxidoreductase reaction in intact tissues. The enzyme activity was assayed by use of radiolabeled estrone and estradiol-17β as substrates. Estrone formation from estradiol-17β was demonstrated in all tissues. The formation of estradiol-17β from estrone was demonstrated in most tissues, however, it was barely detected or was undetectable in the glandular stomach, small intestine, cecum, and large intestine. Thus, 17β-hydroxysteroid oxidoreductase activity is expressed in all BALB c mouse organs and tissues. Approximately two-thirds of the tissues and organs examined, including those of the reproductive tracts, favored the conversion of estrone to estradiol-17β rather than the reverse reaction. The results of this study, however, represent qualitative estimates of 17β-hydroxysteroid oxidoreductase activity in BALB c mouse tissues that are uncorrected for conversion to hydroxylated metabolites. These in vitro findings suggest that the 17β-hydroxysteroid oxidoreductase catalyzed reduction of estrone may contribute to the maintenance of physiologic levels of estradiol-17β in estrogen responsive tissues.