1. The reactions of cytochrome omicron in intact cells of aerobically grown Escherichia coli with O2 and CO have been studied at low temperature. 2. Flash photolysis of CO-liganded cells in the presence of O2 and at temperatures between -79 and -102 degrees C results in the oxidation of kinetically heterogeneous beta-type cytochromes (including cytochrome omicron), but not of cytochrome d. 3. The reaction of reduced cytochrome omicron with O2 involves O2 binding to give intermediate(s) with spectral characteristics similar to that of the reduced oxidase-CO complex. Observation in the alpha-region suggests that unexplained ligand dissociation accompanies the initial O2 binding. 4. At temperatures below -98 degrees C, an 'end point' in the reaction is reached; further reaction and oxidation of cytochrome omicron occurs on raising the temperature. 5. There is a linear relationship between the rate of formation of the oxygen compound and the O2 concentration up to 0.5 mM. The second-order constant for its formation (k+1) is 0.91 M-1.S-1 at -101 degrees C. The reaction is not readily reversible, the value of k-1 being 1.4 X 10(-5) S-1 and the kd 1.5 X 10(-5) M. 6. The energy of activation for this reaction at low temperatures is 29.9kJ (7.1 kcal)/mol. 7. The reaction with O2 is distinguished from that with CO by the markedly lower velocity and high photolytic reversibility of the latter. 8. Comparisons are drawn between the intermediate(s) in the O2 reaction of cytochrome omicron in E. coli and those identified in other bacteria and in the reaction of cytochrome aa3 with O2.