Reversible inhibition of cytochrome c oxidase (CcOX) by nitric oxide (NO') has potential physiological roles in the regulation of mitochondrial respiration, redox signaling, and apoptosis. However peroxynitrite (ONOO-), an oxidant formed from the reaction of NO. and superoxide, appears mostly detrimental to cell function. This occurs both through direct oxidant reactions and by decreasing the availability of NO. for interacting with CcOX. When isolated CcOX respires with ascorbate as a reducing substrate, the conversion of ONOO- to NO. is observed. It is not known whether this can be ascribed to a direct interaction of the enzyme with ONOO-. In this investigation, the role of ascorbate in this system was examined using polarographic methods to measure NO. production and CcOX activity simultaneously in both the purified enzyme and isolated mitochondria. It was found that ascorbate alone accounts for >90% of the NO. yield from ONOO- in the presence or absence of purified CcOX in turnover. The yield of NO was CcOX-independent but was dependent on ascorbate and ONOO- concentrations and was not affected by metal chelators. Consistent with this, the interaction of ONOO- with CcOX in respiring isolated mitochondria only yielded NO. when ascorbate was also present in the incubation. These observations are discussed in the context of ONOO-/ascorbate reactivity and the interaction of CcOX with reactive nitrogen species.