A series of new 4-(alkylthio)-substituted androstenedione analogues was designed as potential suicide inhibitors of aromatase on the basis of mechanistic considerations on the mode of action of the enzyme. Their synthesis and biological evaluation are described. Among the most interesting are the 4-[(difluoromethyl)thio]-, 4-[(fluoromethyl)thio]-, and 4-[(chloromethyl)thio]androstenediones 12, 13, and 14 with respective IC50's of 2.7, 0.8, and 0.94 microM. Compound 12 was a reversible inhibitor of aromatase while compounds 13 and 14 displayed time-dependent kinetics of inhibition with respective KI's and half-times of inactivation of 30 nM and 3.75 min for 13 and 30 nM and 3 min for 14. The inhibition of aromatase by 14 was NADPH-dependent, and was protected by the presence of substrate (0.5-1 microM), while beta-mercaptoethanol (0.5 mM) failed to protect the enzyme from inactivation. Dialysis failed to reactivate aromatase previously inactivated by 14. The mechanistic implications of these findings are discussed.