Hypothalamic prepro-orexin as well as pituitary and adrenal orexin receptors are gender-specifically expressed. To assess the regulation by gonadal steroids, we investigated the effect of 17β-estradiol in female and of testosterone in male rats on prepro-orexin and orexin receptor mRNA expression. Rats were either sham-operated or gonadectomized and subsequently treated with placebo, 17β-estradiol, or testosterone for 21 d. Tissue mRNA levels of prepro-orexin, orexin type-1 (OX1), and orexin type-2 (OX2) receptors were measured using quantitative real-time RT-PCR. In female rats, pituitary OX1 receptor mRNA levels were increased 12-fold after ovariectomy compared with sham-operated rats. The increase of pituitary OX1 receptor mRNA was inhibited by treatment with 17β-estradiol. Adrenal mRNA levels of OX2 receptors in ovariectomized rats were increased 2-fold compared with sham-operated rats and were also reduced by treatment with 17β-estradiol. In male rats, orchidectomy increased the mRNA levels of pituitary OX1 receptors compared with sham-operated rats. In contrast, adrenal OX2 receptor mRNA was reduced after orchidectomy. Testosterone treatment reversed the effect of orchidectomy on pituitary OX1 and adrenal OX2 receptors. In the hypothalamus, no differences were found in the mRNA levels of prepro-orexin, OX1, and OX2 receptors between sham-operated, placebotreated, and steroid-treated female or male rats. Our results indicate that gonadal steroids differentially regulate pituitary OX1 receptors and adrenal OX2 receptors in male and female rats and may contribute to specific sex-dependent neuroendocrine and endocrine actions of orexins.