Bois and Estabrook have described a cycle of absorbance changes occurring in inner membrane preparations of heart mitochondria which is initiated by addition of reduced nicotinamide-adenine dinucleotide (NADH) and may be followed at 470 mμ with 500 mμ as the reference wavelength. There is a rapid bleaching followed by an incomplete, slower return of color. The time of the cycle, the rate of reoxidation, and the amount of irreversibly bleached chromophore are increased by rotenone. These observations are extended in the present study and interpreted in terms of the oxidation-reduction of components associated with the respiratory chain-linked NADH dehydrogenase. In particular, the effect of specifically and unspecifically bound rotenone and piericidin A, of mercurials, and of a combination of mercurials and piericidin on the various parameters of the cycle have been investigated. The findings provide strong support for the applicability of this experimental procedure, using the particular wavelength pair mentioned, to the study of the redox cycle of the dehydrogenase as an enzyme unit.