Myoglobin (Mb) binds diatomic ligands, like O-2, CO, and NO, in a cavity that is only transiently accessible. Crystallography and molecular simulations show that the ligands can migrate through an extensive network of transiently connected cavities but disagree on the locations and occupancy of internal hydration sites. Here, we use water H-2 and O-17 magnetic relaxation dispersion (MRD) to characterize the internal water molecules in Mb under physiological conditions. We find that equine carbonmonoxy Mb contains 4.5 +/- 1.0 ordered internal water molecules with a mean survival time of 5.6 +/- 0.5 mu s at 25 degrees C. The likely locations of these water molecules are the four polar hydration sites, including one of the xenon-binding cavities, that are fully occupied in all high-resolution crystal structures of equine Mb. The finding that water escapes from these sites, located 17-31 angstrom apart in the protein, on the same mu s time scale suggests a global exchange mechanism. We propose that this mechanism involves transient penetration of the protein by H-bonded water chains. Such a mechanism could play a functional role by eliminating trapped ligands. In addition, the MRD results indicate that 2 or 3 of the 11 histidine residues of equine Mb undergo intramolecular hydrogen exchange on a mu s time scale.