Two series of 6 beta- and 6 alpha-substituted androst-4-ene-3,17-diones (5 and 6) were synthesized as aromatase inhibitors to gain insights of structure-activity relationships of varying substituents (methyl, ethyl, n-propyl, isopropyl, n-butyl, phenyl, benzyl, vinyl, and ethynyl) to the inhibitory activity. All of the inhibitors synthesized prevented human placental aromatase in a competitive manner. The inhibition activities of all the 6-n-alkylated steroids 5a-d and 6a-d (Ki = 1.4-12 nM) as well as the 6 beta-vinyl (5h), 6 alpha-benzyl (6g), and 6-methylene (10) compounds (Ki = 5.1, 10, and 4.9 nM, respectively) were very powerful whereas those of the 6-isopropyl (5e and 6e), 6-phenyl (5f and 6f), 6 beta-benzyl (5g), and 6 beta-ethynyl (5i) steroids, having a bulky or polar substituent, were relatively weak. The 6 beta-ethyl derivative 5b was the most potent inhibitor among those synthesized. Inhibitors 5a, 5f, 5h, 5i, 6b, and 10 did not cause a time-dependent inactivation of aromatase. The 6 beta-alkyl steroids essentially had higher affinity for the enzyme than the corresponding 6 alpha-isomers, whereas the opposite relation was observed in a series of the aryl steroids. These results along with molecular modeling with the PM3 method clearly indicate that aromatase has a hydrophobic binding pocket with a limited accessible volume in the active site in the region corresponding to the beta-side rather than the alpha-side of the C-6 position of the substrate.