We have investigated the serine protease gamma-chymotrypsin (gamma-CT) in three different solvation environments using molecular dynamics simulations. These solvation environments include the following : (1) gamma-CT taken from the crystal structure of Yennawar et al. (Biochemistry 1994, 33, 7326-7336) with seven surface bound hexane molecules and 50 bound "essential" water molecules all immersed in 1109 hexane molecules (simulation labeled CT); (2) gamma-CT taken from Yennawar et al. and solvated with 50 "essential" water molecules and immersed in 1107 hexane molecules (simulation labeled CTWAT); and (3) gamma-CT taken from Yennawar et al. and solvated with a monolayer of 444 water molecules and immersed in 931 hexane molecules (simulation labeled CTMONO), From these trajectories we found that the placement of bound water molecules and the amount of hydration of the protein in the simulated structure had an effect on the protein flexibility as indicated by changes in the RMS deviation. The radius of gyration value was similar for the three systems, indicating no significant unfolding or denaturation. Hydrophobic residues were found to have increased solvent accessible surface area (SASA), while hydrophilic residues experienced a decrease in SASA in the CT and CTWAT simulations. No hexane diffusion into the protein interior was found, with the exception of one bound hexane site in the CT simulation. The secondary structure analysis of the active site indicates that the active site structure was retained in all three simulations. We also found that intramolecular forces (i.e., hydrogen bonding) that stabilize proteins are stronger in the CT and CTWAT systems, as shown by the increase in the number of stable hydrogen bonds found. Net ion pair interactions and reduced ratio of surface area to volume of the protein also contributed to the stability of the protein in anhydrous organic media.