Two stereoisomers of natural rotenone (5'alpha-epirotenone and 5'beta-epirotenone) were synthesized to identify the stereochemical factor of rotenone required for the inhibition and also to probe the structure of the rotenone binding site. The inhibitory action of the stereoisomers was compared with that of rotenone using NADH-ubiquinone reductases from bovine heart submitochondrial particles (SMP), potato tubers (Solanum tuberosum L.) SMP and Escherichia coli (GR19N) membranes. With respect to bovine heart SMP, it was found that the bent form of rotenone is essential for the activity. The modification of the E-ring moiety also affected both the inhibitory potency and the pattern of inhibition. These results indicated that the rotenone-binding site recognizes the whole molecular structure (or shape) of rotenone in a strict sense. Rotenone and 5'beta-epirotenone inhibited the NADH-ubiquinone reductase of bovine heart SMP in a noncompetitive manner against exogenous quinones. In contrast, the inhibition pattern of 5'alpha-epirotenone varied from noncompetitive to competitive as the concentration of quinone increased. These results suggest that rotenone binds close to, but not at a site identical to, the location for ubiquinone in the ubiquinone-catalytic reaction site, whereas the 5'alpha-epirotenone-binding site overlaps that for ubiquinone due to a structural modification of E-ring moiety. Furthermore, the complex inhibition pattern of 5'alpha-epirotenone suggests that there are two quinone-binding sites in NADH-ubiquinone reductase. In contrast, the order of the inhibitory potencies of the three inhibitors with proton-pumping NADH-ubiquinone reductase of potato SMP was the same as that observed for the bovine enzyme. This suggests that the structure of rotenone-binding sites (or ubiquinone-binding sites) of these enzymes are similar. It was further demonstrated that 5'alpha-epirotenone inhibits quinone binding to both proton-pumping and non-proton-pumping NADH-ubiquinone reductases of potato SMP in a competitive manner. With respect to the proton-pumping NADH-ubiquinone reductase of the E. coli membrane, the sensitivity of the enzyme to the inhibitor was remarkably decreased and the difference in the inhibitory potencies of the three inhibitors became ambiguous. In addition, the inhibition pattern of the three inhibitors was competitive against quinone. These results indicated that, contrary to the mammalian enzyme, only part of the rotenone molecule is recognized by the quinone-binding site of this enzyme.