This project is the development of a tool for general use in crystallographic refinement of protein structure and in studies of protein structure by simulation. Given the coordinates of the protein, Dowser seeks out internal cavities, and determines the hydrophilicity of each cavity in terms of the energy of placing a water molecule in the cavity, and reports coordinates of water molecules placed in the most hydrophilic cavities. This project is an offshoot of a collaborative research project, and is being continued as a Technical R&D project. The following describes Dowser's scheme of operation, with a pdb file of the protein as only input. step 1: Identify buried cavities inside proteins: Calculate solvent-accessible surface. Points on solvent accessible surface are tested to determine if they are external or internal. Internal surfaces are further classified as open or closed. The program prints out the gross volume of the protein and the volume of the closed cavities by default. The coordinates of the cavity surfaces are output to a file in pdb format and thus can be displayed by a molecular graphics program, such as Rasmol. step 2: Exhaustive search: One at a time, trial waters molecules are placed on the internal surfaces at a certain density (typically 3 per ?2). For each, the energy is minimized by translating and rotating the water molecule. (For this step, polar hydrogen atoms are included in the coordinates. The protein coordinates do not change.) step 3: Select the best candidates: The energy-minimized solvent molecules are sorted according to energy and those with energy below a pre-set threshold (typically -10 or -12 kcal/mol) are retained as likely buried water molecules. In case water molecules overlap into a cluster, the one with lowest energy is selected first, and any molecule with which it overlaps is eliminated. This elimination step is repeated until the last low-energy position has been processed. As an example, when applied to the cyclophylin molecule Dowser did not miss any water molecule discovered by crystallographic refinement. However Dowser found a new potential water binding site which is energetically favorable (-16 kcal/mol); this site is under further investigation.