The first picosecond infrared vibrational echo experiments on a protein, myoglobin-CO, are described. These vibrational dephasing experiments examine the influence of protein dynamics on the CO ligand bound to the active site of the protein at physiologically relevant temperatures. The experiments were performed with a mid-IR free electron laser tuned to the CO stretch mode at 1935 cm(-1). The vibrational echo results are combined with infrared pump-probe measurements of the CO vibrational lifetime to yield the homogenous pure dephasing, the Fourier transform of the homogeneous line width with the lifetime contribution removed. The measurements were made from 60 to 300 K. The results show that the CO vibrational spectrum is inhomogeneously broadened, even at room temperature. Above the glycerol/water solvent’s glass transition temperature, similar to 185 K, the temperature dependence can he fit as an activated process with Delta E approximate to 1000 cm(-1). Below 185 K, the pure dephasing displays a power law temperature dependence, T-1.3. This temperature dependence is reminiscent of that associated with the properties of low-temperature glasses (<5 K) but is observed at much higher temperatures. A two-level system model of protein dynamics is considered. The nature of the temperature dependence and the mechanism of the coupling of the protein fluctuations to the CO vibrational transition energy are discussed.