Objective: To evaluate the effects of tissue culture on the viability and development of follicles in frozen-thawed human fetal ovarian tissue before transplantation into severe combined immunodeficient (SCID) mice and to determine the optimal duration of pretransplant tissue culture. Design: Experimental prospective study. Setting: Animal center and reproductive laboratories in university hospitals. Intervention(s): Frozen-thawed human fetal ovarian tissue samples from 20-week-old abortuses were randomly divided into four groups and cultured in vitro for 0, 3, 6, or 9 days before being xenografted into kidney capsules of bilaterally oophorectomized severe combined immunodeficient (SCID) mice. Grafts were removed 16 weeks after transplantation. Histological analysis and assessment of proliferative cell nuclear antigen (PCNA) expression levels were used to evaluate the survival and development of follicles. Result(s): The proportion of growing follicles was significantly increased in groups cultured before transplantation as compared with the noncultured group. Sixteen weeks after transplantation, the number of follicles in the cultured grafts was higher than that in the noncultured grafts. Grafts cultured for 6 or 9 days showed higher proportions of preantral and antral follicles than grafts cultured for 0 or 3 days. PCNA immunohistochemical analysis indicated that follicle cells were in a proliferative state after culture and transplantation. Conclusion(s): The viability and development of human fetal follicles may be improved by pretransplant tissue culture. The optimal culture duration before transplantation of fetal ovarian tissue is 6 days. © 2010 American Society for Reproductive Medicine.