Abstract A single species of tryptophan-59 formylated cytochrome c with a half-reduction potential of 0.085 ± 0.01 V at pH 7.0 was used to study its catalytic and functional properties. The spectral properties of the modified cytochrome show that the 6th ligand position is open to reaction with azide, cyanide, and carbon monoxide. Formylated cytochrome c binds to cytochrome c depleted rat liver and pigeon heart mitochondria with the precise stoichiometry of two modified cytochrome c molecules per molecule of cytochrome a ( K D of approx 0.1 μ m ). Formylated cytochrome c was reducible by ascorbate and was readily oxidized by cytochrome c oxidase. The apparent K m value of the oxidase for the formylated cytochrome c was six times higher than for the native cytochrome and the apparent V was smaller. Formylated cytochrome c does not restore the oxygen uptake in C-depleted mitochondria but inhibits, in a competitive manner, the oxygen uptake induced by the addition of native cytochrome c . Formylated cytochrome c was inactive in the reaction with mitochondrial NADH-cytochrome c reductase but was able to accept electrons through the microsomal NADPH-cytochrome c reductase.