The C-O stretching frequencies of fully reduced carbonmonoxy cytochrome ba3, a newly discovered terminal oxidase of the bacterium Thermus thermophilus (Zimmermann, B.H., Nitsche, C.I., Fee, J.A., Rusnak, F., and Münck, E. (1988) Proc. Natl. Acad. Sci. U.S. A. 85, 5779-5783), are studied by Fourier transform infrared spectroscopy. Multiple C-O frequencies are observed in the Fourier transform infrared spectra, indicating the presence of discrete interconverting conformers of the enzyme. Upon photolysis, the CO is shown to migrate exclusively to CuB+. Above 200 K, the CO returns to the heme a3 by a thermal process which follows simple first-order kinetics. The rate of the reaction was studied from 205 to 230 K and at 300 K, yielding the activation parameters delta H = 14.9 kcal/mol and delta S = -5 cal/mol/K. These are compared with previously determined activation parameters for CO recombination in mitochondrial cytochrome aa3 preparations (Fiamingo, F.G., Altschuld, R.A., Moh, P.P., and Alben, J.O. (1982) J. Biol. Chem. 257, 1639-1650). We report the novel finding that CO remains bound to CuB+ at room temperature during continuous photolysis of cytochrome ba3, and we conjecture on the possible interference of copper-bound CO in "flow-flash" and "triple-trap" studies of cytochrome c oxidases.