Replacement of the 21-methyl group of 20β-hydroxypregn-4-en-3-one with an ethoxyacetylene (EtO—C≡C—) group yields a compound that is an excellent substrate (pH 7.4, Km = 2.3 μM, Vmax= 4.6 nmol min-1 μg-1) for the Streptomyces hydrogenans NAD(H)-dependent 20/3-hydroxysteroid dehydrogenase (EC 18.104.22.168). The enzyme-generated ethoxyacetylenic ketone product is a potent inactivator of the enzyme. Gel filtration chromatography of enzyme inactivated with radiolabeled steroid demonstrates that covalent modification of the enzyme has occurred. Both NAD and NADH retard the rate of inactivation, suggesting that only free enzyme is susceptible to covalent modification. Consequently, enzymatically formed ethoxyacetylenic ketone does not react with the enzyme while it is part of the ternary complex. Moreover, the kinetically preferred release of this reactive ketone prior to NADH release assures that enzyme inactivation occurs only when released ketone subsequently encounters free enzyme. Kinetic analysis of inactivations carried out with chemically prepared ethoxyacetylenic ketone and enzyme at pH 7.4 and 9.2 yields bimolecular rate constants for the inactivation process of 1.15 x 104 L mol-1 s-1 and 6.94 x 104 L mol-1 s-1, respectively. This bimolecular reaction is faster than the bimolecular reaction of the ethoxyacetylenic ketone with either glutathione, mercaptoethanol, or dithiothreitol. Thus, complete inactivation by ketone generated from 5 μM alcohol and 5 μM NAD occurs in 30 min at pH 7.4 in the presence of 1 mM glutathione.