The synthesis and biological evaluation of 3-alkyl-substituted 3-(4-aminophenyl)piperidine-2,6-diones as inhibitors of estrogen biosynthesis are described [H (1), methyl (2), ethyl (3), n-propyl (4), isopropyl (5), n-butyl (6), isobutyl (7), sec-butyl (8), n-pentyl (9), isopentyl (10), 2-methylbutyl (11), sec-pentyl (12), n-hexyl (13), n-heptyl (14)]. In vitro compounds 4-14 showed a stronger inhibition of human placental aromatase compared to aminoglutethimide (AG, compound 3), which recently has become used for the treatment of hormone-dependent breast cancer. The most active derivative, compound 10, showed a 93-fold stronger inhibition than AG. With the exception of 5, 7, and 8, all other compounds exhibited similar or decreased inhibition of bovine adrenal desmolase compared to AG. Compounds 4 and 6-12 showed a stronger inhibition of the plasma estradiol concentration of pregnant mare serum gonadotropin (PMSG) primed Sprague-Dawley (SD) rats compared to the parent compound. Compounds 4, 6-8, 10, and 12 inhibited the testosterone-stimulated tumor growth of ovariectomized 9,10-dimethyl-1,2-benzanthracene (DMBA) tumor-bearing SD rats more strongly than AG. Being stronger and more selective inhibitors of the estrogen biosynthesis than AG, some of the newly developed derivatives of AG might be better candidates for the treatment of the hormone-dependent human breast cancer.