To determine an effective in vitro culture method of rabbit immature ovarian follicles, preantral follicles (200 to 299 µm in diameter) were cultured by the whole follicle culture method and by the open type culture method. The use of tuna matrix collagen (MC) as a medium material to maintain 3-dimensional structure of the follicles was also investigated for the open type culture method. In the whole follicle culture method, the diameter of the preantral follicles increased from 252.8 ± 2.7 µm to 395.6 ± 9.6 µm. However, culture for oocyte maturation was not possible because most of the oocytes had degenerated. In the open culture method, in which oocyte-granulosa cell complexes (OGs) collected from preantral follicles were cultured, the rates of the oocytes that resumed meiosis were 1.6%, 5.4%, and 73.7% in 0, 0.3, and 3 mg/ml of MC supplementation, respectively. Morphologically, the addition of MC was found to show a beneficial effect for maintenance of the three-dimensional structure of the follicles. In conclusion, open type culture with MC supplementation seems to be a most appropriate culture method for rabbit immature follicles.