A method for the measurement of water that is strongly held by lysozyme is described. This water is slowly removed by vacuum drying of lyophilized lysozyme and can be titrated with the Karl Fischer reagent. Drying curves were obtained by mechanical pumping (moderate vacuum) and diffusion pumping (high vacuum) at 20°, 10°, 0°, -10°, and -20°C. About 23 water molecules per lysozyme molecule are at least moderately held by the protein. These water molecules fall into several classes. Three to four of them are quite strongly held and may correspond to the three buried water molecules observed in the x-ray analysis of lysozyme structure. The presence of tri-N-acetylglucosamine in the lysozyme active cleft has no effect on drying at 0°C. The method shows promise of being generally applicable to the measurement of small amounts of water which are strongly held by biological structures.