Lipid-binding proteins Sequester amphiphilic molecules in a large internal : cavity occupied by similar to 30 water molecules, some of which are displaced by the ligand. The role; of these internal water molecules in lipid binding and release is not understood. We use magnetic relaxation dispersion (MRD) to directly monitor internal-water dynamics in apo and palmitate-bound rat intestinal fatty acid-binding protein (rIFABP). Specifically, we record the water H-2 and O-17 MRD profiles of the apo and hobo forms of rIFABP in solution or immobilized by covalent cross-links. A global analysis of this extensive data set identifies three internal-water classes with mean survival times of similar to 1 ns, similar to 100 ns, and similar to 6 mu s. We associate the two longer time scales with conformational fluctuations of the gap between beta-strands D and E (similar to 6 mu s) and of the portal at the helix-capped end of the beta-barrel (similar to 100 ns). These fluctuations limit the exchange rates of a few highly ordered structural water molecules but not the dissociation rate of the fatty acid. The remaining 90% (apo) or 70% (hobo) of cavity waters exchange among internal hydration sites on a time scale of similar to 1 ns but exhibit substantial orientational order, particularly in the holo form.