The steady-state kinetics of the NADH dehydrogenase activities of the mitochondrial NADH-ubiquinone oxidoreductase in the presence of one-electron acceptors, ferricyanide and hexammineruthenium(III), were studied. Similar to ferricyanide, hexammineruthenium was found to be an efficient electron acceptor for the enzyme in inside-out submitochondrial particles and isolated Complex I, but not in intact mitochondria. Qualitatively the same results were obtained using submitochondrial particles or isolated Complex I. Both hexammineruthenium(III) and ferricyanide reduction was rotenone-insensitive and showed no stimulation by the uncouplers in tightly coupled submitochondrial particles. In contrast to the NADH-ferricyanide oxidoreductase reaction which exhibits a double substrate inhibition behaviour, no inhibition of the reaction by either NADH or the electron acceptor was revealed in the NADH-hexammineruthenium(III) reductase reaction. The double-reciprocal plots 1/v vs. 1/[NADH] at various hexammineruthenium(III) concentrations gave a series of straight lines intercepting in the third quadrant, thus supporting the mechanism of the overall reaction in which the reduced enzyme-NAD+ complex is oxidized by the electron acceptor before NAD+ dissociation. The apparent KsNADH values equal to 1 x 10(-5) and 4 x 10(-5) M for submitochondrial particles and Complex I, respectively (27 degrees C, pH 8.0), were determined from the secondary KmNADH vs. V (at different acceptor concentrations) plot. The Ki values for the competitive inhibition of NADH oxidation by NAD+ were 1 x 10(-3) M and 2 x 10(-3) M for the respective enzyme preparations. The results obtained suggest that hexammineruthenium(III) interacts with the NADH-ubiquinone oxidoreductase at a single reaction site different from that for fericyanide.