AB - The two steroidal 17 beta-oxiranes, spiro-17 beta-oxiranyl-delta 4-androsten-3-one (4 beta) and spiro-17 beta-oxiranylestra-1,3,5(10),6,8-pentaene-3-ol (5 beta) are active site-directed irreversible inhibitors of bacterial delta 5-3-ketosteroid isomerase. For each inhibitor, a stoichiometry of one molecule of steroid to one enzyme subunit was found. The inhibited enzyme was denatured and subjected to digestion by trypsin. The tryptic maps show two distinct steroid-bound peptides for both 4 beta- and 5 beta-inhibited isomerase. In each case, the two modified peptides are derived from residues 14 to 45 of the isomerase. Each of the steroid-bound peptides of the 4 beta-inhibited enzyme was subjected to further proteolytic digestion and the site of steroid attachment was found to be Asp-38 in each of the inactivation products. These results are interpreted to indicate that "backwards binding" is an important feature of the binding of steroids to delta 5-3-ketosteroid isomerase.