Mullerian inhibiting substance (MIS) in the adult rat ovary can be detected by immunohistochemistry in the granulosa cells of growing preantral follicles and in the cumulus oophorus and periluminal granulosa cells of antral follicles in estrus, metestrus, diestrus, and proestrus. Neither the corpus luteum nor atreitic follicles stained for MIS. During proestrus, dramatic changes occurred in the large preovulatory antral follicles, which early in the day manifest intense MIS-specific staining in the granulosa cells located close to the oocyte, however, at 2300 h, just before ovulation when the germinal vesicle was extruded to indicate resumed meiotic division, MIS staining waned. When the morphology of the late preovulatory stage was created experimentally in 26-day-old immature ovaries by stimulating 48 h earlier with hCG, the intense staining of the granulosa cells surrounding the oocytes from untreated ovaries was lost in the cumulus cells of such hCG-treated animals. This temporal pattern of MIS staining and the prior demonstration that MIS could inhibit in vitro meiosis of oocytes from untreated immature rats suggest that this regressor, well characterized in the fetal testis, might function in the ovary as a regulator of oocyte maturation and follicular development during the adult reproductive cycle.