To characterize further the active site of human placental estradiol 17 beta-dehydrogenase (EC 1.1.1.62), we have synthesized 2-bromoacetamidoestrone methyl ether. The affinity-labeling steroid is a substrate for the homogeneous enzyme. It inactivates the enzyme in a time-dependent, irreversible manner which follows pseudo-first order kinetics. Further, inactivation conducted with varying steroid concentration displays saturation kinetics. When 1.7 x 10(-6) M enzyme is inactivated by 2.6 x 10(-4) M 2-bromoacetamidoestrone methyl ether, the presence of an equimolar concentration of estradiol or 5.2 x 10(-4) M concentrations of NAD+, NADP+, or NADPH markedly slow the rate of inactivation. Bromoacetate (2.6 x 10(-4) M) does not inactivate the enzyme. After inactivation with 2-bromo[2'-14C]acetamidoestrone methyl ether, amino acid analysis reveals carboxymethylated derivatives of cysteine, histidine, and lysine containing 65, 25, and 8%, respectively, of the total incorporated carboxymethyl groups. The presence of estradiol, NADP+, or NADPH clearly inhibits alkylation of cysteinyl and histidyl residues and slows the rate of enzyme inactivation. Protection of these residues by both estradiol and NADPH suggests that they may actually be in the cofactor region of the active site, that this region is close to the steroid A-ring, and that binding of cofactor, by physical interposition, denies the reagent-bearing steroid access to these residues.