A number of synthetic steroid analogues which contain conjugated acetylenic ketones, namely 10β-(1-oxoprop-2-ynyl) estr-4-ene-3,17-dione (I). 5,10-secoestr-4-yne-3,10,17-trione (II), 17β-hydroxy-5,10-secoestr-4-yne-3, 10-dione (III) and 17β-(1-oxoprop-2-ynyl)androst-4-en-3-one (IV), irreversibly inactivate C19- and C21-Δ5-3-ketosteroid isomerase (EC 126.96.36.199) activities of beef adrenal cortex microsomes in a time dependent manner. For each compound examined, the maximum rate of alkylation of C19-Δ5-3-ketosteroid isomerase activity is different to that observed for the alkylation of C21-Δ5-3-ketosteroid isomerase activity. At saturation, half lives range from 90-2760 s corresponding to k+ 2 values (the limiting rate constant for inactivation) of 0.25-7.6 × 10-3 · -1. Under suitable conditions, compounds (I-IV) also behave as competitive inhibitors of these enzymatic reactions. With androst-5-ene-3,17-dione as substrate the Ki values were 0.080 μM for (IV), 7.5μM for (II), 30 μM for (I) and 53 μM for (III), whereas with pregn-5-ene-3,20-dione as substrate the corresponding values were 0.013, 3.0, 7.0 and 15 μM, respectively. These findings suggest that the C19- and C21-Δ5-3-ketosteroid isomerase(s) of bovine adrenal cortex microsomes may be separate enzymes and that the latter has a more hydrophobic binding site. Comparison of the apparent bimolecular rate constants (k+2/Ki) for the inactivation of both C19- and C21-Δ5-3-ketosteroid isomerase activities gives the following order of efficiency for inactivation (IV) > (II) > (I) > (III). The high efficiency of compound (IV) as an inactivator is a reflection of its extremely low Ki and it is suggested that this compound may be a transition state analogue. For the Δ5-3-ketosteroid isomerase from Pseudomonas testosteroni. the order of efficiency of inactivation given by k+2/Ki is quite different, with (I) > (II) > (IV) > (III).