In order to further characterize the active site of fetal lamb 3β, 20α-Hydroxysteroid Oxidoreductase, we have synthesized the radioactive 16α-bromoacetoxyprogesterone and 5α-dihydrotestosterone 17-bromoacetate as affinity labeling reagents. Both of them are the irreversible, active-site-directed and competitive inhibitors of the enzyme. When the concentration of the enzyme is 1 μmol/L, inhibitor is 100 μmol/L, the inactivation reaction followed pseudo-first-order kinetics with a t_0.5=75 min (16α-BAP) and t_0.5=480min (5α-DTB). When substrate progesterone or 5α-dihydrotestosterone was present in the incubation mixture, the speed of inactivation of the enzyme was slowed down. The labeled enzyme was hydrolyzed by hydrochloric acid, through amino acid analysis, the labeled histidine was identified. The histidine at active site may play important role in the oxidoreductive reaction.