In order to examine a possible role of the pituitary gland and uterine tissue in the formation of 13,14-dihydroprostaglandin F2 alpha (13,14H2-PGF2 alpha) in rat ovary, hypophysectomized and hysterectomized rats were used. Gonadotropins stimulated the formation of 13,14H2-PGF2 alpha from prostaglandin F2 alpha (PGF2 alpha) and 13,14-dihydro-15-keto-PGF2 alpha (15KD-PGF2 alpha) in the ovarian homogenate of hypophysectomized and hysterectomized rats as well as in intact rats. Ovarian steroids, estradiol and progesterone, reduced the formation of 13,14H2-PGF2 alpha in the ovarian homogenate of intact rats. However, in hysterectomized and hypophysectomized rats, its formation was not affected by ovarian steroids. On the other hand, when pregnant mare serum gonadotropin (PMS) and estradiol were simultaneously administered to intact and hypophysectomized rats, the formation of 13,14H2-PGF2 alpha in the ovary showed a tendency to be increased as compared with that after treatment with PMS alone. These results not only suggest that the formation of 13,14H2-PGF2 alpha in rat ovary is regulated by gonadotropins and ovarian steroids, but also that uterine tissue may take part in the process of its formation.