A synthetic quinone, 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT), inhibits electron transfer reactions in the cytochrome b-c1 segment of the mitochondrial respiratory chain. Addition of UHDBT to isolated succinate-cytochrome c reductase complex has effects on reduction of the cytochromes b and c1 by succinate similar to those which result from removal of the iron-sulfur protein from the b-c1 complex. Thus, UHDBT inhibits reduction of cytochrome c1 by succinate and, if antimycin is added before succinate, UHDBT inhibits reduction of cytochrome b in addition to c1. UHDBT increases the midpoint potential of the iron-sulfur protein of the b-c1 complex from +280 to +350 mV at pH 7.2. The inhibitor also shifts the gx peak in the EPR spectrum of the iron-sulfur protein from g = 1.80 to 1.76 and shifts the gz peak from g = 2.02 to 2.03. It causes only a slight shift in the central gy = 1.90 signal. The efficacy of inhibition of cytochrome c reductase activity of isolated reductase complex by UHDBT appears to depend on the oxidation-reduction poise of some component(s) in the b-c1 complex. Inhibition is decreased and there is an extensive lag in the onset of inhibition under conditions favoring oxidation of the b-c1 complex; inhibition increases and the lag is eliminated under conditions favoring reduction of the b-c1 complex. The titer for inhibition of cytochrome c reductase activity of isolated reductase complex is one UHDBT per b-c1 complex. With reductase complex from which the iron-sulfur protein of the b-c1 complex is reversibly resolved, the titer for inhibition is proportional to the amount of iron-sulfur protein reconstituted to the complex. These results suggest that UHDBT inhibits mitochondrial respiration by binding to the iron-sulfur protein of the b-c1 complex, possibly at a site which is otherwise involved in binding ubiquinone, and that this binding is enhanced when the iron-sulfur protein is reduced.