To access the production of a novel uterine luminal fluid 250?kD protein (ULF 250) during the various phases of the estrous cycle in relation to estrogen concentration, and to validate that the production and secretion of ULF 250 are regulated by estradiol and progesterone Methods An immunohistochemical method was used to localize ULF 250 in rat uteri during each phase of the estrous cycle, and in uteri of ovariectomized rats treated with estradiol and progesterone Results Positive immunostaining of ULF 250 occurred in the epithelial cells of the uterus at all phases of the estrous cycle; whereas the stroma was immunonegative During the proestrus phase of the cycle, the glandular epithelial cells and glandular luminal content were stained strongly During the estrus phase of the cycle, intense staining occurred in the glandular and uterine luminal epithelial cells, including the luminal content of the glands In the metestrus phase of the cycle, only uterine epithelial cells were stained; during the diestrus phase, intense staining of the secreted contents of the uterine cavity and the glandular lumen occurred The distribution of ULF 250 in the uteri of ovariectomized female rats treated with estradiol alone and estradiol plus progesterone were examined In both groups, intense staining of the glandular luminal epithelial cells of the uterine endometrium occurred; in the estradiol treated animals, only the luminal contents were stained The present findings suggest that progesterone inhibits the secretion of ULF 250 that is stimulated by estradiol Conclusions ULF 250 is produced by the glandular and luminal epithelial cells of the uterine endometrium and fluctuates with the phases of the estrous cycle Its production is stimulated by estradiol and its secretion is regulated by progesterone To access the production of a novel uterine luminal fluid 250® kD protein (ULF 250) during the various phases of the estrous cycle in relation to estrogen concentration, and to validate that the production and secretion of ULF 250 are regulated by estradiol and progesterone Methods An immunohistochemical method was used to localize ULF 250 in rat uteri during each phase of the estrous cycle, and in uteri of ovariectomized rats treated with estradiol and progesterone Results Positive immunostaining of ULF 250 occurred in the epithelial cells of the uterus at all phases of the estrous cycle; and the stroma was immunonegative During the proestrus phase of the cycle, the glandular epithelial cells and glandular luminal content were stained strongly during the estrus phase of the cycle, and the intense media occurred in the glandular and uterine luminal epithelial cells, including the luminal content of the glands In the metestrus phase of the cycle, only uterine epithelial ce lls were stained; during the diestrus phase, intense staining of the secreted contents of the uterine cavity and the glandular lumen occurred The distribution of ULF 250 in the uteri of ovariectomized female rats treated with estradiol alone and estradiol plus progesterone were examined In both groups, intense staining of the glandular luminal epithelial cells of the uterine endometrium occurred; in the estradiol treated animals, only the luminal contents were stained The present findings suggest that progesterone inhibits the secretion of ULF 250 that is stimulated by estradiol Conclusions ULF 250 is produced by the glandular and luminal epithelial cells of the uterine endometrium and fluctuates with the phases of the estrous cycle Its production is stimulated by estradiol and its secretion is regulated by progesterone