The pituitary and hypothalamic content of dynorphin was determined by radioimmunoassay and characterized by high-performance liquid chromatography (HPLC) in adult female Sprague-Dawley rats, intact and ovariectomized with and without estrogen treatment. Animals were given estradiol benzoate, or vehicle (oil) by six daily intramuscular injections. Anterior pituitary content of immunoreactive (ir)-dynorphin in ovariectomized rats was approximately twice that of intact animals, and consisted of a single HPLC peak co-eluting with dynorphin 32. Administration of estradiol benzoate (0.06–6 µg/day) caused a marked decrease of ir-dynorphin in the anterior lobe of castrate female rats, with a half-maximal effect at 0.2 µg/day; levels were restored to those seen in intact animals with 6 µg estradiol benzoate per day, an effect which was not influenced by concomitant administration of progesterone (1 mg/day), or bromocriptine (100 µg/day). In the hypothalamus and neuro-intermediate lobe multiple peaks of immunoreactive dynorphin were seen, coeluting with dynorphin A 1–8, dynorphin A 1–17 and dynorphin 32. Neither castration nor estrogen treatment altered ir-dynorphin content in these tissues. These findings suggest that the ovary exerts a specific modulating influence on AP ir-dynorphin in the rat, and that in addition this inhibition appears to be mediated by ovarian estrogen.