The NADH-ubiquinone reductase activity of the respiratory chains of several organisms was inhibited by capsaicin and dihydrocapsaicin, which are the pungent principles of red pepper. This inhibition was correlated with the presence of an energy transducing site in this segment of the respiratory chain. Where the NADH-quinone oxidoreductase segment involved an energy coupling site (e.g., in Paracoccus denitrificans, Escherichia coli, and Thermus thermophilus HB-8 membranes and bovine heart mitochondria), capsaicin acted as an inhibitor of ubiquinone reduction by NADH. In contrast, where this energy coupling site was absent (e.g., in Saccharomyces cerevisiae mitochondria and Bacillus subtilis membranes), there was no inhibition of NADH-ubiquinone reductase activity by capsaicin. The capsaicin inhibition of Paracoccus membranes was reversed by washing the membranes with medium containing bovine serum albumin. In the E. coli and Paracoccus membranes and bovine submitochondrial particles, capsaicin acted as a noncompetitive inhibitor for ubiquinone-1 at lower concentrations of ubiquinone-1 (less than 20 microM) and as a competitive inhibitor at higher concentrations of ubiquinone-1 (greater than 50 microM). In addition, the concentrations of capsaicin required for 50% inhibition of NADH oxidase activity of bovine submitochondrial particles were increased when ubiquinone-10 was added to the particles. The mechanism by which capsaicin inhibits the energy-transducing NADH-quinone oxidoreductase is discussed.