The synthesis of 3-cycloalkyl-substituted 3-(4-aminophenyl)piperidine-2,6-diones is described [cyclopentyl (1), cyclohexyl (2)]. The enantiomers of 2 were separated either by using HPLC on optically active sorbent or by crystallization of the brucine salt of the phthalamic acid of 2. The absolute configuration of the (+)- and (-)-enantiomers of 2 were assigned as S and R, respectively, by comparing the CD spectra to those of the enantiomers of aminoglutethimide (AG, 3-(4-aminophenyl)-3-ethylpiperidine-2,6-dione). The compounds were tested in vitro for inhibition of human placental aromatase, the cytochrome P450-dependent enzyme which is responsible for the conversion of androgens to estrogens. Compounds 1 and 2 inhibited aromatase by 50% at 1.2 and 0.3 microM, respectively (IC50 AG = 37 microM). According to the findings with AG, the (+)-enantiomer of 2 was responsible for the inhibitory activity, being a 240-fold more potent aromatase inhibitor in vitro than racemic AG. On the other hand, (+)-2 displayed a strongly reduced inhibition of desmolase (cholesterol side-chain cleavage enzyme) compared to AG (relative activity = 0.3). Thus (+)-2 is of interest as a potential drug for the treatment of estrogen-dependent diseases, e.g. mammary tumors.