Dibucaine-HCl inhibited mitochondrial cytochrome c oxidase activity in intact mitochondria with 50% inhibition occurring at 1.1 mM dibucaine-HCl. Dibucaine-HCl did not prevent the reduction of cytochrome oxidase by ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride (TMPD) when measured at 604 nm but prevented 50% of the absorbance change at 445 nm; dithionite reduced the oxidase completely. Dibucaine prevented binding of CO to oxidase reduced with ascorbate plus TMPD by preventing the reduction of cytochrome a3. The midpotenials of cytochrome c and cytochrome oxidase, the visible absorbance wavelength maxima, and the position and intensity of the signals of the EPR spectrum of the oxidase were not affected. Dibucaine-HCl prevented ascorbate plus TMPD-driven reduction of the near infra-red detectable copper center associated with cytochrome a: dithionite subsequently reduced this center. Dibucaine-HCl inhibited cytochrome oxidase activity by interacting between cytochrome a and its associated copper. Since respiration was 8-fold less sensitive in submitochondrial particles, this site of inhibition is on the cytoplasmic side of the membrane.