The β,γ-acetylenic ketosteroids, 5,10-seco-19-norpregn-5-yne-3,-10,20-trione 1 and 5,10-secoestr-5-yne-3,10,17-trione 2 irreversibly inactivate both the C 19- and the C 21-Δ 5-3-ketosteroid isomerase activities of beef adrenal cortex microsomes. At saturating concentrations of inhibitor half-lives of these enzyme activities vary from 45 to 240 s. It is uncertain whether the enzyme generates its own alkylating agent by isomerizing compounds 3 and 4 to the corresponding allenic ketones, namely (4 R )-5,10-seco-19-norpregn-4,5-diene-3,10,20-trione 3 and (4 R )-5,10-secoestra-4,5-diene-3,10,17-trione 4 since these are formed spontaneously in the buffer used to stabilize enzyme activity. In the presence of catalytic quantities of adrenal enzyme compound 4 is a powerful competitive inhibitor for both 5-androstene-3,17-dione (K i 8.0 μM) and 5-pregnene-3,20-dione (K i 3.5 μM) indicating that the eventual alkylation event is active site-directed. The differences in K i values and half-lives for inactivation support ti view that the C 19- and C 21-Δ 5-3-ketosteroid isomerase activities do not reside at the same catalytic site in beef adrenal cortex microsomes.