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The effect of Ca²⁺ on the rate of heme a reduction by dithionite and hexaammineruthenium (RuAm) was studied in the cyanide-complexed bovine cytochrome oxidase (CcO). The rate of heme a reduction is proportional to RuAm concentration below 300 μM with k_v of 0.53 × 10⁶ M⁻¹s⁻¹. Ca²⁺ inhibits the rate of heme a reduction by dithionite by ∼25%. As the reaction speeds up with increased concentrations of RuAm, the inhibition by Ca²⁺ disappears. The inhibition of heme a reduction may contribute to recently described partial inhibition of CcO by Ca²⁺ in the enzymatic assays. The inhibitory effect of Ca²⁺ on heme a reduction indicates that ET through heme a may be coupled to proton movement in the exit part of the proton channel H.