We sought to establish a new in vitro ovulation model using the whole ovaries of the medaka. Ovaries of the fish, which had been acclimated to the usual reproductive conditions (26°C, 14 h light/10 h dark) and which had then been kept at least one day at 30°C, were isolated 2 h before the expected in vivo ovulation time. When the ovaries were cultured in 90% medium 199 solution at 30°C or 36°C, oocytes were liberated with a gradual increase in the ovulation rate at 2 to 5 h of ovulation time. The maximum ovulation rate was ∼45%. Ovulated oocytes were fertilized and subsequently developed into adults. In vitro ovulation of medaka ovaries was inhibited by the addition of metalloproteinase inhibitors to the culture. In this in vitro ovulation model, the holes formed on the follicle layer upon follicle rupture at ovulation were sealed, strongly suggesting the importance of the germinal epithelium in the process. The present study indicates that our new in vitro ovulation model is useful for investigating the role of germinal epithelial cells in the ovulate process of the medaka fish.