Type 3 17beta-hydroxysteroid dehydrogenase (17beta-HSD) is involved in the biosynthesis of the potent androgen testosterone (T), which plays an important role in androgen-sensitive diseases. In an attempt to design compounds to lower the level of T, we designed androsterone (ADT) derivatives substituted at the position 3beta as inhibitors of type 3 17beta-HSD, and then selected the eight most potent ones (compounds 1-8) for additional studies. In an intact cell assay, they inhibited efficiently the conversion of natural substrate 4-androstene-3,17-dione into T, although they were less active in intact cells (IC50 approximately 1 microM) than in homogenated cells (IC50=57-100 nM). A study of the inhibitory potency with four other 17beta-HSDs revealed they were selective, since they do not inhibit reductive types 1, 5 and 7, nor oxidative type 2. Interestingly, they did not show any binding affinity for steroid receptors (androgen, estrogen, glucocorticoid and progestin). Only two inhibitors, 3beta-phenyl-ADT (5) and 3beta-phenylmethyl-ADT (6) showed some proliferative activities on an AR+ cell line and on an ER+ cell line, but their effects were not mediated through the androgen or estrogen receptors. This study identified selective inhibitors of type 3 17beta-HSD acting through a mixed-type inhibition, and devoid of non-suitable androgenic and estrogenic proliferative activities. The more potent inhibitors were 3beta-hexyl-ADT (2), 3beta-cyclohexylethyl-ADT (4) and 3beta-phenylethyl-ADT (7).