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Annexin 1 (ANXA1) is a Ca2+- and phospholipid-binding protein that plays an important role as a mediator of glucocorticoid action in the host-defence and neuroendocrine systems. Sex differences in hypothalamo–pituitary–adrenal (HPA) axis activity are well documented and a number of studies have demonstrated that gonadal steroids act as regulators of HPA activity. The aim of this study was to investigate the effect of ovariectomyand 17β-estradiol replacement, and estrous cycle stage, on anterior pituitary ANXA1 content. The amount of anterior pituitary ANXA1 determined by western blotting varied with estrous cycle stage with a peak at estrus declining to a trough at proestrus. Ovariectomy resulted in a significant (P<0.05) decrease in anterior pituitary ANXA1 content. Administration of 17β-estradiol (1 μg/100 g) significantly (P<0.01) increased anterior pituitary ANXA1 expression in the ovariectomized animals. In contrast, there was no change in pituitary ANXA1 content in response to 17β-estradiol in adrenalectomized and adrenalectomized/ovariectomized rats. Treatment of TtT/GF cells, a folliculo-stellate cell line, with 17β-estradiol (1.8–180 nM) increased ANXA1 mRNA expression and increased the amount of ANXA1 protein externalized in response to a dexamethasone stimulus. These results indicate that 17β-estradiol stimulates ANXA1 expression in the anterior pituitary and in vivo an adrenal factor contributes to the mechanism of action.