Overfishing and environmental factors such as pollution have contributed to the significant decline of fish populations worldwide. Successful cryopreservation of fish gametes and embryos would have important applications in conservation and aquaculture. Although cryopreservation of fish semen has already been achieved successfully, cryopreservation of fish embryos and oocytes remains elusive. Despite the widespread use of vitrification for oocyte cryopreservation in humans and domestic mammals, vitrification of fish oocytes has not been studied to date. This study aimed at developing a cryopreservation protocol for stage III zebrafish ovarian follicles within fragment using vitrification. Vitrification ability of methanol, ethanol, Me 2 SO, propylene glycol and ethylene glycol were tested using a range of concentrations made up in 90% Leibovitz L-15 medium. Solutions were tested for vitrification ability using 0.25 ml plastic straw and a fibreplug™ (CryoLogic Ltd.) loaded by a pipette. The transparent glassy appearance during cooling and warming was used to identify vitrified solution. Based on results obtained from this stage, the vitrification ability of 24 combinations using the mentioned cryoprotectants was studied. Three vitrification solutions (VS) (V6B: 1.5 M methanol + 6.0 M ethylene glycol + 0.5 M sucrose; V1B: 1.5 M methanol + 4.5 M propylene glycol; and V2: 1.5 M methanol + 5.5 M Me 2 SO) were selected for subsequent experiments based on their vitrifying ability. For VS toxicity tests, ovarian follicles were equilibrated in L-15 medium containing 1.5 M methanol for 30 min at room temperature. After equilibration follicles were exposed to VS for 3 min in a stepwise manner. Afterwards the cryoprotectants were gradually removed in three steps and viability was assessed by trypan blue staining. For vitrification procedures, immediately after exposure to VS ovarian follicles were loaded in either plastic straw or fibreplug™ and plunged into liquid nitrogen, being stored for at least 20 min. The warming was performed at 28 °C and cryoprotectants were gradually removed in three steps. Ovarian follicle viability was assessed by three means: trypan blue staining, measurement of ATP level in the follicles and assessment of mitochondrial membrane potential and distribution by using JC-1 molecular probe. Results obtained from toxicity tests showed follicles viability of 74% for V1B; 30% for V2 and 78% for V6B. After vitrification, ovarian follicles presented a viability of 60% when fibreplug and V1B were used, V2 showed viability of 42% (using fibreplug), and using plastic straws V6B solution showed only 2% of viability. [ATP] soon after warming dropped down to 22% (V2) and 7% (V1B) and two hr later the concentration in V2 was 15% and for V1B a very low concentration of 3.5%. The viable follicles in V1B and V2 seemed to be opaque and morphologically intact, when assessed immediately after warming. These results are different to the results described so far in the literature, where zebrafish follicles were observed to become swollen and translucent even during the warming process after being exposed to controlled slow cooling protocols. However, 30 min after warming most of the follicles started to become semi-translucent and lightly swollen. A complete loss of internal structure pattern and also loss of fluorescence indicated follicles with no mitochondrial membrane potential after vitrification.