Abstract: A carefully controlled, multistage freezing and thawing process for the preservation of animal semen, blood, and other biological materials. A variety of diluting substances can be utilized to effectively modify the cell membrane's permeability to water and to initiate and/or alter the process of crystallization to minimize undesirable physio-chemical effects.A sample of semen is diluted with a suitable cryoprotective agent to achieve a final pH of 6.0 to 6.3 at +5.degree.C. The sample is collected at body temperature and cooled slowly to +5.degree.C, held at this temperature for 30 minutes or longer, followed by a rapid decrease in temperature to a nominal -4.degree.C, or a temperature slightly below the freezing point of the diluent. The sample is held at this temperature for a period of 1 to 8 minutes to allow adjustment and stabilization of the temperature induced changes in pH and associated alterations in membrane permeability and osmotic pressure.