The kinetics of water penetration and escape in cytochrome c (cyt c) is studied by molecular dynamics (MD) simulations at various temperatures. Water molecules that penetrate the protein interior during the course of an MD simulation are identified by monitoring the number of water molecules in the first coordination shell (within 3.5 Å) of each water molecule in the system. Water molecules in the interior of cyt c have 0 -3 water molecules in their first hydration shell and this coordination number persists for extended periods of time. At T ؍ 300 K we identify over 200 events in which water molecules penetrate the protein and reside inside for at least 5 picoseconds (ps) within a 1.5 nanoseconds (ns) time period. Twenty-seven (27) water molecules reside for at least 300 ps, 17 water molecules reside in the protein interior for times longer than 500 ps, and two interior water molecules do not escape; at T ؍ 360 K one water molecule does not escape; at 430 K all water molecules exchange. Some of the internal water molecules show mean square displacements (MSD) of 1 Å 2 characteristic of structural waters. Others show MSD as large as 12 Å 2 , suggesting that some of these water molecules occupy transient cavities and diffuse extensively within the protein. Motions of protein-bound water molecules are rotationally hindered, but show large librations. Analysis of the kinetics of water escape in terms of a survival time correlation function shows a power law behavior in time that can be interpreted in terms of a broad distribution of energy barriers, relative to B T, for water exchange. At T ؍ 300 K estimates of the roughness of the activation energy distribution is 4 -10 kJ/mol (2-4 B T). Activation enthalpies for water escape are 6 -23 kJ/mol. The difference in activation entropies between fast exchanging (0.01 ns) and slow exchanging (0.1-1 ns) water molecules is ؊27 J/K/mol. Dunitz (Science 1997;264:670.) has estimated the maximum entropy loss of a water molecule due to binding to be 28 J/K/mol. Therefore, our results suggest that the entropy of interior water molecules is similar to entropy of bulk water.