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This paper presents the Danish 10-year experience (1999-2009) with cryopreservation (n=386) and autotransplantation of ovarian tissue (n=18). Before applying the technique to humans, the method was thoroughly tested and validated. The cryoprotectant solution was chosen after histological evaluation of mouse and human ovarian tissue after freezing with four different combinations of cryoprotectants. Viability was confirmed by transplantation of frozen-thawed human ovarian tissue (n=49) to oophorectomized Nude mice. Viability after transport of fresh tissue 4-5h prior to freezing had previously been validated. Overnight transport of fresh ovarian tissue prior to cryopreservation was evaluated when human ovarian tissue was kept on ice for 20h and then cryopreserved. The thawed ovarian tissue was transplanted to an oophorectomized Nude mouse and histology confirmed viability. In Denmark 12 women have received a total of 18 autotransplantations of ovarian tissue. All women resumed ovarian function and three healthy babies were born to two women. In both women, the tissue was transported on ice for 4-5h prior to cryopreservation. Ovarian tissue cryopreservation is an important method for fertility preservation; however, before applying the method clinically, each laboratory should perform thorough validation of their technique.