Ovarian steroids have previously been shown to regulate the hypothalamic content of β-endorphin (βEP) and its release into hypophyseal portal blood. Although the hypothalamic content of βEP in cycling female rats was unchanged by ovariectomy, chronic treatment of ovariectomized rats with estradiol lowered hypothalamic βEP levels. In this study, the hypothalamic content of βEP was compared in male and cycling female rats, and the effects of orchiectomy and testosterone replacement on hypothalamic βEP were examined. The j8EP content of the medial basal hypothalamus (MBH) was significantly higher in female rats compared to that in males of either the same weight (175–200 g) or the same age (65 days; P < 0.025). When male rats were studied 4 weeks after castration, the β-EP content of the MBH increased from a value of 2100 ± 103 fmol in the controls to 2680 ±126 fmol (P < 0.005). The hypothalamic βEP content in the castrated males was similar to that in the intact females (2700 ± 158 fmol). The increase in hypothalamic 0EP induced by castration was blocked by testosterone replacement. When orchiectomized animals were treated for 4 weeks with Silastic capsules filled with testosterone, there was a significant fall in the hypothalamic content of j8EP compared to that in the unreplaced animals. βEP fell from 3180 ± 115 to 2033 ± 53 fmol in the MBH (P < 0.001), from 1693 ± 122 to 934 ± 80 fmol in the anterior hypothalamus (P < 0.001), and from 148 ± 26 to 90.3 ±11 fmol in the median eminence (P < 0.05). Testosterone replacement was also associated with a significant decline in the hypothalamic content of corticotropinlike intermediate lobe peptide and aMSH. Corticotropin-like intermediate lobe peptide fell from 2400 ± 53 to 1560 ± 84 fmol in the MBH (P < 0.001) and from 1200 ± 74 to 805 ± 94 fmol in the anterior hylpothalamus (P < 0.01). aMSH fell from 1660 ± 162 to 884 ± 75 fmol in the MBH (P < 0.001) and from 823 ± 106 to 544 ± 92 fmol in the anterior hypothalamus (P < 0.05). Thus, testosterone, as well as estradiol, affects the hypothalamic content of several proopiomelanocortin-derived peptides. The effect on brain peptide content, however, depends on whether the steroids are secreted relatively constantly, as in the male, or fluctuate, as in the cycling female. (Endocrinology119: 19–24, 1986)