Estradiol 17 beta-hydroxysteroid dehydrogenase (17 beta HSD) mediates the interconversion of estrone and estradiol in endocrine-responsive tissues such as the breast. The control of 17 beta HSD expression by all-trans-retinoic acid (RA) in T47D breast cancer cells was examined using a specific 17 beta HSD complementary DNA probe. Two main 17 beta HSD messenger RNA (mRNA) transcripts of 2.2 and 1.3 kilobases (kb) were detected, of which only the 1.3-kb mRNA was regulated. RA increased expression of the 17 beta HSD 1.3-kb mRNA in a dose- and time-dependent manner, and the increased expression of this mRNA by RA was inhibited by a 10-fold excess of a RA antagonist Ro 41-5253. Insulin-like-growth factor-I, interleukin-1, and estradiol, previously shown to increase 17 beta HSD activity in breast cancer cells, had little effect on 17 beta HSD gene expression. To relate the effect of increased 17 beta HSD 1.3-kb mRNA expression to 17 beta HSD activity, the conversion of estrone to estradiol (reductive) and that of estradiol to estrone (oxidative) were measured in intact T47D cell monolayers. Whereas RA increased 17 beta HSD reductive activity, it had no effect on oxidative activity. The addition of excess NAD increased 17 beta HSD oxidative activity in control and RA-treated cells, but the addition of NADH had no effect on 17 beta HSD reductive activity. These results suggest that the increased expression of the 17 beta HSD 1.3-kb mRNA induced by RA is associated with an increase in 17 beta HSD reductive activity, but that endogenous cofactor levels may determine the direction in which this enzyme acts in T47D cells.