The normal-mode refinement of X-ray crystallographic data opened a new possibility to analyze the mean-square displacements in a protein molecule. A comparison of the X-ray structure of myoglobin at several temperatures with Mo¨ssbauer data is performed. In the low-temperature regime below 180 K the iron mean-square displacements obtained by Mo¨ssbauer spectroscopy are in good agreement with a normal-mode analysis. The X-ray mean-square displacements at the position of the iron, after the motion originated from the external degrees of freedom are subtracted, have practically the same temperature dependence as those from Mo¨ssbauer spectroscopy. The difference between the X-ray mean-square displacements and those predicted by normal-mode analysis measures the distribution of molecules into conformational substates. Above 180 K the Mo¨ssbauer effect indicates fluctuations between conformational substates. The relaxation from a Fe(III) conformation to a Fe(II) conformation is shown for superoxide dismutase ofPropionibacterium shermanii. ©1999 American Institute of Physics.