The oxygenerated form of cytochrome o can be readily observed in whole cell suspensions of Vitreoscilla because it is the predominant steady state form of the cytochrome in respiring cells. When oxygen is introduced to an anaerobic suspension of cells, the steady state is established immediately and absorption maxima are observed at 578, 545, and 425 nm in difference spectra using ammonium persulfate-oxidized cells as reference. The oxygenerated form of cytochrome o is identified as the predominant steady state species during turnover of the cytochrome in vivo from the close coincidence of these spectra with those obtained using purified cytochrome. When the dissolved oxygen is consumed, the spectrum of the oxygenated form is replaced by the spectrum of the reduced form. Glutamate increased the respiration of these cells and decreased the duration of the steady state. Spectra of the oxygenated form of cytochrome o could also be obtained by using starved cells as reference, i.e. (untreated cells) - (starved cells) difference spectra. These results implicate the oxygenated form of cytochrome o as an active intermediate in terminal electron transport of these cells. In the presence of 1 mM cyanide, which inhibits the respiration of Vitreoscilla, the duration of the steady state was increased and the steady state spectrum was altered, showing that turnover of the cytochrome was inhibitied and formation of the oxygenated form wsa decreased. All these results suggest that the cyclic changes of cytochrome o in terminal respiration involve the oxidized, reduced, and oxygenated forms.