An increase in the ovarian secretion of progesterone occurring on the afternoon of the day following ovulation was observed even in the rats hypophysectomized after ovulation, confirming the automaticity of newly formed corpora lutea secreting progesterone independently of pituitary function. Either estrogen (estradiol, estradiol benzoate) or prolactin (NIH-P-S8) given shortly after ovulation prolonged the rise of ovarian progesterone secretion as well as the duration of diestrous phase in vaginal smears. When rats were hypophysectomized preceding the hormone treatment, estrogen was not effective but prolactin was effective to cause luteotrophic action (prolongation of increased progesterone secretion), indicating that estrogen acts indirectly on the ovaries by stimulating the secretion of luteotrophic substance from the pituitary. 20α-Hydroxypregn-4-en-3-one secretion, which is known to be almost parallel with progesterone secretion in the normal diestrous stage, was lowered during the period when exogenous estrogen produced luteotrophic action to sustain the increase of progesterone secretion.