In the pituitary, CRH-binding protein (CRH-BP) neutralizes the ACTH-releasing activity of CRH. Because sexual dimorphisms exist at multiple levels of the hypothalamic-pituitary-adrenal axis, these studies examined expression of pituitary CRH-BP in the male and female mouse pituitary. Ribonuclease protection assays and (125)I-CRH cross-linking assays demonstrate greater expression of pituitary CRH-BP in female than male mice. Normalized CRH-BP mRNA levels in female mice are 2.58 times greater at proestrus than diestrus. Ovariectomy reduces pituitary CRH-BP mRNA levels to 11% of sham-ovariectomy control levels, and estradiol benzoate treatment restores CRH-BP mRNA to control levels. These data suggest that estrogen positively regulates pituitary CRH-BP. Dual in situ hybridization analysis reveals that CRH-BP expression increases significantly in proopiomelanocortin-expressing cells at proestrus, compared with metestrus (P = 0.003), suggesting that CRH-BP expression is estrogen regulated in corticotropes. Further studies reveal that approximately 80% of the CRH-BP transcripts in the proestrus mouse pituitary localize to prolactin-expressing cells, a novel site for CRH-BP expression. CRH-BP mRNA levels increase significantly at proestrus, compared with metestrus in prolactin-positive cells (P < 0.0001). This robust, estrogen-regulated expression of CRH-BP in lactotropes in female mice suggests that the pituitary is an important site for interactions between the hypothalamic-pituitary-adrenal axis and other endocrine systems.