In vitro conversion in human endometrial tissue of Org OD 14 [17 alpha-hydroxy-7 alpha-methyl-19-norpregn-5(10)-en-20-yn-3-one, a 3-keto-delta 5-10-19-nortestosterone derivative structurally related to norethynodrel] to its 4-ene isomer was demonstrated and measured spectrophotometrically and by chromatographic separation of the labeled metabolite from the tritiated precursor. The endometrial isomerase catalyzing this conversion is the 3 beta-hydroxy-steroid dehydrogenase/isomerase (3 beta HSD/isomerase), detected by Western blotting as a 42 kDa band, as confirmed by the inhibition of Org OD 14 isomerization with an antibody against this enzyme. The endometrial isomerase activity was found to be higher in secretory than in proliferative tissue and to be influenced by progestins, as suggested by the small but significant increase in activity resulting from exposure of proliferative endometrium to medroxyprogesterone acetate under organotypic culture conditions. In addition to the expected physiologic importance of endometrial 3 beta HSD/isomerase in the local metabolism of circulating steroids of adrenal origin, its presence in the endometrium is likely to have pharmacologic relevance, as illustrated by the local conversion of Org OD 14 to the 4-ene isomer, a metabolite with higher progestagenic and lower estrogenic potencies than those of its precursor. The local, tissue-specific, modification of the precursor would yield intracellular concentration ratios of Org OD 14 to 4-ene isomer in the endometrium significantly lower than those in blood. As a result, the estrogenic effects of Org OD 14 or of its 3-hydroxy metabolites on endometrial cell proliferation are minimized by the local formation of the progestagenic 4-ene isomer. This is a favorable feature of Org OD 14 since it selectively prevents undesirable proliferative stimulation of the endometrium in postmenopausal users while preserving its beneficial effects on other tissues, including bone.