The primary intermediate in the reaction of oxygen with cytochrome c oxidase was generated by photodissociating carbon monoxide in a continuous flow rapid mixing apparatus. The presence of the primary intermediate was confirmed by a comparison of the iron-dioxygen stretching frequency with that obtained in the reaction of oxygen with the mixed-valence enzyme. For both of these preparations, the Fe-O2 stretching mode is detected at 568 cm-1, the same frequency as that found in oxyhemoglobin and oxymyoglobin. These data illustrate that the primary intermediate may be generated and detected at room temperature in the fully reduced enzyme and that the oxidation state of cytochrome a does not affect the structure of the iron-dioxygen complex. By following the changes in the intensity of the Fe-O2 stretching mode in the resonance Raman spectrum as a function of time, the first-order rate constant for the decay of the primary intermediate was found to be 3.5 x 10(4) s-1 (t1/2 = 20 microseconds).