The binding sites of rotenone, piericidin A, and Amytal in the reduced nicotinamide adenine dinucleotide oxidase chain of heart have been studied with the aid of rotenone-6a-14C. Binding of rotenone continues in a linear manner beyond the point of maximal inhibition of respiration, indicating that rotenone is tightly bound not only at the specific site in the NADH dehydrogenase segment of the respiratory chain but also at other sites in submitochondrial particles. Unspecific binding is reduced by treatment of the particles with rotenone in the presence of bovine serum albumin (BSA) and is further minimized by successive washing with BSA, with only minimal removal of rotenone from the specific site. Rotenone-14C is not removed from the specific site by denaturation or proteolysis but is largely removed by repeated extractions with anhydrous acetone, which results in recovery primarily of rotenone with only small amounts of a less inhibitory oxidation product. Substantial reversal of the inhibition of respiration occurs on repeated washing of the inhibited particles with BSA. Thus, contrary to expectations, under usual assay conditions rotenone is not a specific, stoichiometric, and irreversible inhibitor. Studies involving preincubation of the electron transport particle with various unlabeled inhibitors, of widely varying chemical type and inhibitory potency, prior to addition of rotenone-14C for determination of its subsequent binding, provide strong evidence that it is possible to distinguish between the portion of rotenone-14C that is specifically bound and the portion that is unspecifically bound. Piericidin A, Amytal, and a number of rotenoids appear to react at the same site as rotenone because they compete with rotenone-14C for binding at the specific site, and the competition with rotenone is proportional to the inhibitory power of the compounds tested and not to their concentration or chemical nature. Although the molar ratio of rotenone bound at the specific site to the NADH dehydrogenase content in particles approximates unity, the binding is not on NADH dehydrogenase itself, since extraction of the enzyme from phosphorylating or non-phosphorylating particles labeled with rotenone-14C results in nearly complete separation of rotenone-14C from the enzyme. Qualitative and quantitative differences are evident in the binding of rotenone to soluble NADH-coenzyme Q reductase as compared to submitochondrial particles. Although the soluble reductase, extracted with acid-ethanol from particles inhibited with rotenone-14C, is fully active and contains little or no rotenone-14C, it may be inhibited again on further addition of rotenone.