To investigate whether estradiol can act within the ovary to induce luteolysis in the rhesus monkey, 100 mug estradiol-17beta were injected on one of days 2, 3, or 4 after the preovulatory LH peak into one of the following sites: the stroma of the ovary containing the developing corpus luteum, the stroma of the contralateral ovary, or sc. When estradiol was injected into the ovary containing the corpus luteum, the functional life span of the corpus luteum was shortened, reflected by a premature decline in circulating progesterone to levels characteristic of the follicular phase of the cycle and an early onset of menstruation. When estradiol was injected either sc or into the ovary contralateral to that containing the corpus luteum, the life span of the corpus luteum was not shortened. The differing responses could not be attributed to differing rates of efflux of estradiol from the various injection sites; patterns and levels of estradiol in peripheral serum were essentially the same regardless of injection site. Furthermore, the premature regression of the corpus luteum was not a consequence of surgical trauma associated with the intra-ovarian injection procedure, nor could it be mimicked by another ovarian hormone, progesterone. These findings lead to the conclusion that estrogens can induce functional luteolysis in the rhesus monkey by acting directly within the ovary containing the corpus luteum.