In previtellogenic oocytes of the freshwater teleost, Oryzias latipes, the yolk nucleus is a prominent structure in the cytoplasm. The process by which the animal-vegetal (A-V) axis is established in these oocytes was observed by focusing on the yolk nucleus, granulosa cells and attaching filaments using light and electron microscopy. Prior to appearance of the rudiments of attaching filaments a special area was recognized near the yolk nucleus of early stage III oocytes by gathering of granulosa cells with irregular-shaped nuclei, The rudiments of attaching filaments clearly appeared in the area where granulosa cells gathered. The area with a cluster of granulosa cells and the rudiments of attaching filaments corresponded to the position of the yolk nucleus. In late stage III, the future vegetal pole area (VPA) was easily recognizable by the presence of the rudiments of attaching filaments. The present results suggest that the vegetal pole may be primarily determined by the random position of the yolk nucleus and established by reciprocal interactions between the cortical cytoplasm containing the yolk nucleus and its surrounding granulosa cells. It was postulated that the animal pole is determined by differentiation of the micropylar cell from a granulosa cell and by locomotion of granulosa cells around the axis connecting the centers of the germinal vesicle and the yolk nucleus before the initiation of vitellogenesis.