Multinuclear NMR spectroscopy has been used to study water bound to subtilisin Carlsberg suspended in tetrahydrofuran (THF), with the water itself employed as a probe of the hydration layer＇s physicochemical and dynamic characteristics. The presence of the enzyme did not affect the intensity, chemical shift or linewidth of water (up to 8% v/v) added to THF, as measured by O-17- and H-2-NMR. This finding suggests that hydration of subtilisin can be described by a three-state model that includes tightly bound, loosely bound, and free water. Solid-state H-2-NMR spectra of enzyme-bound D2O support the existence of a non-exchanging population of tightly bound water. An important implication is that the loosely-bound water is the same as free water from an NMR viewpoint. This loosely bound water must also be the water responsible for the large increase in catalytic activity observed in previous hydration studies.