The rate at which equine and macaque ovarian tissue sections are first cooled from has a significant effect on the measured water transport when the tissues are subsequently frozen in solutions of glycerol, dimethylsulfoxide (DMSO), or ethylene glycol (EG). To determine whether the response of ovarian tissues is altered if they are suspended in mixtures of cryoprotective agents (CPAs), rather than in solutions of a single CPA, we have now measured the subzero water transport from ovarian tissues that were suspended in mixtures of DMSO and EG. Sections of freshly collected equine and macaque ovaries were suspended either in a mixture of EG plus DMSO (equivalent to a mixture of of EG and DMSO) or in a solution of only DMSO or only EG. The tissue sections were cooled from and then frozen to subzero temperatures at . As the tissues were being frozen, a shape-independent differential scanning calorimeter technique was used to measure water loss from the tissues and, consequently, the best fit membrane permeability parameters ( and ) of ovarian tissues during freezing. In the mixture of , the respective values of and for equine tissue first cooled at between and before being frozen were and . The corresponding and values for equine tissue suspended in DMSO were and ; in EG, the values were and , respectively. For macaque ovarian tissues suspended in the mixture of , the respective values of and were and . Similarly, the corresponding and values for macaque tissue suspended in DMSO were and ; in EG, the values were and . The parameters for both equine and macaque tissue samples suspended in the mixture and first cooled at between and were very similar to the corresponding values for samples cooled at . In contrast, the membrane parameters of equine and macaque samples first cooled at in single-component solutions were significantly different from the corresponding values for samples cooled at . These results show that the membrane properties of ovarian cells from two species are different, and that the membrane properties are significantly affected both by the solution in which the tissue is suspended and by the rate at which the tissue is cooled from before being frozen. These observations suggest that these variables ought to be considered in the derivation of methods to cryopreserve ovarian tissues.