Cryosurgery for treating prostate cancer is hampered by an incomplete understanding of the mechanisms whereby tissue destruction is achieved during freezing. The two known biophysical mechanisms of injury, intracellular ice formation and cellular dehydration injury (solute effects), have not been quantified within tumor tissue. Freeze substitution microscopy and a differential scanning calorimeter (DSC) were used to quantify freeze-induced dehydration within Dunning AT-1 rat prostate tumor tissue. Stereological analysis of histological tumor sections was used to obtain the initial cellular (Vo), interstitial, and vascular volumes of the AT-1 tumor tissue. A Boyle-van't Hoff (BVH) plot was then constructed by examining freeze substituted micrographs of equilibrium cooled tissue slices to obtain the osmotically inactive cell volume, Vb = 0.25Vo. Obtaining dynamic cellular water transport information from the freeze substitution microscopy data proved difficult because of the artifact added by the high interstitial volume ( ∼ 35%). Since the DSC technique does not suffer from this artifact, a model of water transport was fit to the DSC water transport data at 5°, 10° and 20°C/min to obtain the combined best fit membrane permeability parameters of the embedded AT-1 tumor cells, assuming either a Krogh cylinder geometry or a spherical cell geometry. Numerical simulations were also performed to generate conservative estimates of intracellular ice volume (IIV) in the tumor tissue at various cooling rates typical of those experienced during cryosurgery ( ≤ 100°C/min). Water transport data in tumor systems with significant interstitial spaces can be obtained by using histology and the low-temperature microscopy methods to obtain the initial and final tissue cell volumes, respectively, and the DSC technique to obtain the dynamic volume changes during freezing.