To gain insight into the spatial nature of the androstenedione (AD) binding (active) site of aromatase in relation to the catalytic function of the enzyme, we synthesized 2,2-dimethylAD (4), 2beta- and 2alpha-methylADs (5 and 6), 19-oxygenated derivatives of compounds 4 and 6, and 2-methyleneAD (17), and we then tested their inhibitory activity as well as their aromatase reaction (aromatization for 2-methyl and 2-methylene analogs or 19-oxygenation for 2,2-dimethyl steroids) with human placental aromatase. 2-Methyl and 2-methylene steroids 5, 6, and 17 were good competitive inhibitors of aromatase (K(i)=22-68nM), but less effective compared to the 2,2-dimethyl analog 4 (K(i)=8.8nM), indicating that a combination of 2beta- and 2alpha-methyl moieties is essential for the formation of a thermodynamically stable inhibitor-aromatase complex. A series of 2alpha-methyl steroids were good substrates for aromatase, whereas 2beta-methyl steroid 5 was an extremely poor substrate, and a series of 2,2-dimethyl steroids did not serve as substrate, suggesting that a 2beta-methyl moiety of the 2,2-dimethyl and 2beta-methyl steroids would prevent the aromatase reaction probably due to steric hindrance in each case. The 2-methylene compound 17 was also aromatized to produce 2-methylestrogen with a low conversion rate where the 1,4-diene structure may have been created before the C(10)-C(19) bond cleavage. Kinetic analysis of the aromatization of androgens revealed that a good substrate was not essentially a good inhibitor for aromatase.