Rat liver 3α-hydroxysteroid dehydrogenase (3α-HSD, EC 22.214.171.124) inactivates circulating androgens, progestins, and glucocorticoids. 3α-HSD is a member of the aldo-keto reductase superfamily, and the X-ray structure of the apoenzyme shows the presence of an (α/β)8 barrel [Hoog, S. S., Pawlowski, J. E., Alzari, P. M., Penning, T. M., & Lewis, M. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 2517–2521]. As yet, a three-dimensional structure of the ternary complex E·NADPH·steroid is unavailable. To identify regions of the enzyme involved in steroid hormone recognition, we have employed mechanism-based inactivators and site-directed mutagenesis. (3RS)-1,10-Seco-5α-estr-1-yne-3,17β-diol (1) and (17RS)-17-hydroxy-14,15-secoandrost-4-en-15-yn-3-one (3) are secosteroids which contain latent Michael acceptors (α,β-unsaturated alcohols) at opposite ends of the steroid nucleus (at the C-3 and C-17 positions, respectively). It was found that compounds 1 and 3 inactivated 3α-HSD only in the presence of NAD+. The requirement for cofactor implies that 1 and 3 are oxidized to the corresponding α,β-unsaturated ketones for inactivation to occur. Chemically prepared 17β-hydroxy-1,10-seco-5α-estr-1-yn-3-one (2) and 14,15-secoandrost-4-en-15-yne-3,17-dione (4), the presumed products of 1 and 3 oxidation, behaved as stoichiometric inactivators of 3α-HSD. In the presence and absence of NAD+, 2 and 4 inactivated >50% of the enzyme in 10 s or less. These results provide evidence for the backward binding of partial steroid substrates and their turnover to reactive acetylenic ketones which alkylate 3α-HSD. Affinity-labeling studies with (bromoacetoxy)steroids have previously identified Cys-170, Cys-217, and Cys-242 as sites of contact for steroid hormones. The crystal structure shows that only Cys-217 resides in the core of the barrel near the presumptive steroid binding site. To determine whether Cys-217 is the reactive nucleophile alkylated by the secosteroids, 1–4 were used to inactivate the following mutants: C170A, C242A, and C217A. The first two mutants demonstrated inactivation kinetics similar to that of native 3α-HSD. The C217A mutant, however, was highly resistant to inactivation by 1–4 and supports the role of Cys-217 in inactivation by the secosteroids. These data are rationalized by molecular modeling.