Background: This study examined the primary effect of selected cryoprotective agents (CPAs) on the meiotic spindles of human oocytes during cooling. Methods: Fresh metaphase II oocytes (n=26) donated from patients undergoing IVF treatment were analyzed via Polscope. In experiment one, 16 oocytes with visible spindle at 37°C were cooled to 20°C and rewarmed to 37°C to test the spindle response to cooling. They were then cooled to 20°C, 10°C, 0°C and rewarmed to 37°C after having been equilibrated with 1.5 M 1,2-propanediol (PROH), 1.5 M dimethyl sulfoxide (DMSO), 1.5 M ethylene glycol (EG) or 10 μM taxol at 37°C. In experiment two, 10 oocytes without visible spindles at 37°C were cooled to 20°C and then equilibrated with PROH, EG and taxol at 20°C. Spindle images were recorded at each temperature. Results: Meiotic spindles remained visible or became more distinct during cooling to 20°C, 10°C and 0°C when equilibrated with PROH, EG, DMSO and Taxol. Without these agents, meiotic spindles of the same oocytes disappeared after cooling to 20°C. Conclusion: The primary effect of PROH, EG and DMSO on the meiotic spindle is to stabilize and protect it against low temperature disassembly. A higher equilibration temperature (≥33°C) for oocyte freezing is recommended.