Adult female Sprague-Dawley rats were killed at different stages of a 4-day estrous cycle, and ovaries and anterior pituitaries examined for content of immunoreactive β-endorphin by RIA and for localization by indirect immunofluorescence. Two anti-β-endorphin antisera, both recognizing different antigenic determinants of human-β-endorphin, showed intense immunfluorescence staining of cells localized predominantly in ovarian corpora lutea. At proestrus, both large and small luteal cells stained positively but only the large luteal cells were immunofluorescence positive at other stages of the estrous cycle. In addition, less intense staining of granulosa cells was occasionally observed in secondary and antral follicles; scattered cells in the interstitium were also weakly positive. In contrast, cells of primordial and primary follicles, and those of theca tissue were consistently negative. Ovarian levels of immunoreactive β-endorphin were found to be lowest at estrus (2.1 ± 0.18 ng/g; n = 8, mean ± SE) and significantly raised in stepwise manner over metestrus and diestrus to a peak (∼4 × estrous levels) at proestrus; in contrast, immunoreactive β-endorphin content of anterior pituitaries remained unaltered during the same period. Sephadex G-50 gel chromatography of ovarian extracts revealed three distinct peaks of immunoreactive β-endorphin, a minor peak in the void volume, and two major peaks of unequal size eluting at mol wt approximately 11.5K and approximately 3.5K. The major species of low molecular weight immunoreactive β-endorphin on reverse phase HPLC was β-endorphin1–31. We conclude from the findings that, in adult rat ovaries, luteal, granulosa, and interstitial cells are responsible for the production of immunoreactive β-endorphin and that this production, being related to the estrous cycle, is presumably under the direct or indirect influence of gonadotropins.