Rat ovarian 20 alpha-hydroxysteroid dehydrogenase was shown to be effectively inhibited by adenosine derivatives, nicotinamide derivatives, NADP analogs, N-alkylammonium chlorides, and carboxylic acids through coenzyme-competitive inhibition studies. Multiple inhibition analysis was used to demonstrate either simultaneous binding of inhibitors that interact with different regions of the NADP-binding site or mutual exclusion of inhibitors that interact with the same region on the enzyme. The results of these studies demonstrated that the 2'-phosphate, the pyrophosphate, and the positively charged ring nitrogen are important features of the coenzyme structure in binding to the coenzyme-binding site of the enzyme. In addition, the presence of a hydrophobic region near the NADP-binding site was indicated by positive chainlength effects observed in the binding of nicotinamide derivatives, alkylammonium chlorides, and carboxylic acids.