We have studied the temperature dependence of the IR stretch bands of carbon monoxide (CO) in the Xe 4 internal cavity of myoglobin mutant L29W-S108L at cryogenic temperatures. Pronounced changes of band areas and positions were analyzed quantitatively by using a simple dynamic model in which CO rotation in the cavity is constrained by a static potential. The librational dynamics of the CO causes a decrease of the total spectral area. A strong local electric field splits the CO stretch absorption into a doublet, indicating that CO can assume opposite orientations in the cavity. With increasing temperature, the two peaks approach each other, because the average angle of the CO with respect to the electric field increases. A combined classical and quantum-mechanical analysis precisely reproduces the observed temperature dependencies of both spectral area and peak shifts. It yields the height of the energy barrier between the two wells associated with opposite CO orientations, V0 approximately 2 kJ/mol, and the frequency of oscillation within a well, omega approximately 25 cm(-1). The electric field in the protein cavity was estimated as 10 MV/cm.