SUMMARY: Viability of rat ovarian tissue frozen and thawed in vitro was assessed by its capacity to form an endocrinologically active autograft, and variables associated with the treatment have been studied. Other neutral solutes tested were not as effective as glycerol in protecting against freezing damage, and 15% (v/v) glycerol was more effective than 10% and much more effective than 5%. The duration of soaking before slow freezing can be reduced with advantage from 1 hr to 15 min. One-stage rapid freezing to −79° C was very damaging to the tissue as compared with slow freezing, but two-stage rapid freezing, recently described as being effective with bull spermatozoa, gave intermediate results. Tissue frozen in glycerol-saline deteriorates rapidly after thawing if left at room temperature, but transference to glycerol horse serum after thawing largely preserves viability. Some implications of this result are discussed.