Objective: In vitro culture and maturation of immature ovarian follicles is an alternative for preserving the fertility potential of young persons using cryopreserved ovarian tissue. The aim of this study was to compare the effect of the mechanical and enzymatic isolation of follicles on the subsequent follicular and oocyte development. Materials and Methods: Preantral follicles were isolated from immature NMRI mice ovaries either mechanically (group M) or enzymatically (group E) and cultured for 10 days. Growth survival rates of the follicles were assessed during the culture period for both groups. Data were analyzed using the chi-square test. Results: After 2 days of culture basement membrane disruption had occurred in 36.7% of group M and 100% group E (p< 0.05). At the end of culture period the survival rate was higher in group M than in group E (74.3 and 63.55 respectively). Antral formation was observed in 29.5% of surviving follicles in group M and 43.1% of group E (p< 0.05). Conclusion: The results indicated the mechanical isolation produces less damage to the preantral follicles than enzymatic isolation and could therefore produce better quality follicles.