To gain insight into the mechanism for irreversible inactivation of aromatase by 6β-bromoandrostenedione (1), one of the earliest discovered suicide substrates, in relation to the catalytic function of the enzyme, the 2,2-dimethyl derivative of compound 1, steroid 4, and its 6α-isomer 5, as well as 2-methyl-1,4-diene steroid 8 and its 6α-bromide 10, were synthesized. All of the steroids inhibited aromatase activity in human placental microsomes with apparent K_i's ranging between 10 and 81 nM. The 2,2-dimethyl-6β- and 6α-bromo steroids 4 and 5 were extremely powerful inhibitors (K_i: 14 and 10 nM, respectively), but these two did not cause a time-dependent inactivation of aromatase in the presence of NADPH; in contrast, the 2-methyl-1,4-diene steroids 8 and 10 caused time-dependent inactivation with apparent k_inact of 0.035 and 0.071 min⁻¹, respectively, in a suicide manner. These results indicate that the 2,2-dimethyl function of the 6β-bromide 4 would prevent the inactivation of aromatase caused by inhibitor 1 in a suicide manner, probably through steric activity, whereas the 2-methyl group of steroid 8 did not significantly affect the suicidal inactivation by the parent 1,4-diene steroid, a typical suicide substrate.