Effects of pregnant mare serum gonadotropin and human chorionic gonadotropin on the formation of 13,14-dihydroprostaglandin F2 alpha, a biologically active compound, were investigated in rat ovarian homogenate. The mass number of the compound, which was formed prostaglandin F2 alpha via 13,14-dihydro-15-ketoprostaglandin F2 alpha in rat ovarian homogenate but was not produced in rat homogenate, accorded with that of the authentic 13,14-dihydroprostaglandin F2 alpha by negative ion chemical ionization mass spectrometry. In the present experiment, the radioactivity of [3H]prostaglandin F2 alpha added to ovarian homogenate was decreased linearly and immediately until the incubation time of 10 min. The formation of 13,14-dihydroprostaglandin F2 alpha was increased up to 60 min. The formation of 13,14-dihydroprostaglandin F2 alpha from prostaglandin F2 alpha was markedly increased by pregnant mare serum gonadotropin and human chorionic gonadotropin. However, there was no additive or synergistic effect of these hormones. The formation of 13,14-dihydroprostaglandin F2 alpha from 13,14-dihydro-15-ketoprostaglandin F2 alpha weas also greatly stimulated by pregnant mare serum gonadotropin and human chorionic gonadotropin. The formation of 13,14-dihydro-15-ketoprostaglandin F2 alpha steeply declined until 24 h after treatment with human chorionic gonadotropin in pregnant mare serum gonadotropin-primed rats. In contrast, the formation of 13,14-dihydroprostaglandin F2 alpha was markedly increased until 24 h after human chorionic gonadotropin treatment, and the level was about 2.5-fold higher than that at 0 h, 48 h after injection of pregnant mare serum gonadotropin.