The cytochrome c peroxidase-catalyzed oxidation of yeast iso-1 ferrocytochrome c by hydrogen peroxide can be understood on the basis of a mechanism involving two cytochrome c-binding sites on cytochrome c peroxidase. Values of the equilibrium dissociation constants for both the high- and low-affinity binding sites determined from the steady-state kinetic measurements agree well with published values obtained by vastly different techniques, providing strong support for the two-binding site mechanism. Maximum enzyme turnover via oxidation of cytochrome c bound at the high-affinity site increases from 2 to 860 s-1 as the ionic strength is increased from 0.010 to 0.20 M. Oxidation of yeast iso-1 ferrocytochrome c is faster in the 2:1 complexes of cytochrome c peroxidase compounds I and II in comparison to the 1:1 complexes. The oxidation rates in the 2:1 complex are macroscopic rate constants equal to the sum of the oxidation rates via both the high- and low-affinity sites. The maximum enzyme turnover via the 2:1 complex increases from 1100 to 2700 s-1 over the ionic strength range 0.010-0.070 M.