The interconversion of estrone (E1) and 17 beta-estradiol (E2), androstenedione (4-ene-dione) and testosterone (T), as well as dehydroepiandrosterone and androst-5-ene-3 beta,17 beta-diol is catalyzed by 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD). The enzyme 17 beta-HSD thus plays an essential role in the formation of all active androgens and estrogens in gonadal as well as extragonadal tissues. The present study investigates the tissue distribution of 17 beta-HSD activity in the male and female rat as well as in some human tissues and the distribution of 17 beta-HSD mRNA in some human tissues. Enzymatic activity was measured using 14C-labeled E1, E2, 4-ene-dione and T as substrates. Such enzymatic activity was demonstrated in all 17 rat tissues examined for both androgenic and estrogenic substrates. While the liver had the highest level of 17 beta-HSD activity, low but significant levels of E2 as well as T formation were found in rat brain, heart, pancreas and thymus. The oxidative pathway (E2----E1, T----4-ene-dione) was favored over the reverse reaction in almost all rat tissues while in the human, almost equal rates were found in most of the 15 tissues examined. The widespread distribution of 17 beta-HSD in rat and human tissues clearly indicates the importance of this enzyme in peripheral sex steroid formation or intracrinology.