Testosterone and estradiol have been shown to affect the hypothalamic content of several pro-opiomelanocortin (POMC)-derived peptides in castrated male and female rats, respectively. It was unclear, however, whether the effects of testosterone on hypothalamic POMC were due to conversion by aromatization to estradiol or whether there were independent androgen actions on hypothalamic POMC. In order to answer this question, the effect of treatment with the nonaromitizable androgen 5-α-dihydrotestosterone (DHT) on the concentration of β-endorphin (β-EP) in the medial basal hypothalamus (MBH) was studied in castrated male rats and compared to the effect of treatment with testosterone or estradiol. The concentrations of two other POMC-derived peptides, corticotropin-like intermediate lobe peptide (CLIP) and α-MSH were measured as well. Adult male rats were castrated and received either no treatment or treatment with subcutaneously implanted silastic capsules, containing either DHT, testosterone or estradiol, designed to produce steroid levels in a physiological range. After 4 weeks the mean concentration of β-EP in the MBH of the untreated castrated rats was 1,640 ± 56 fmol/mg protein. This was reduced significantly to 1,184 + 74 fmol/mg protein after DHT treatment (p < 0.001). Similar reductions to 1,340 + 95 and 1,130 ± 85 fmol/mg protein were noted after testosterone and estradiol treatment, respectively. The mean CLIP concentration of 1,870 ± 73 fmol/mg protein in the untreated animals fell to 1,390 ± 95 after DHT (p < 0.001) compared to 1,520 ± 105 and 1,260 ± 101 after testosterone and estradiol treatment, respectively. α-MSH was similarly reduced from 911 ± 42 fmol/mg protein to 723 ± 54 after DHT (p < 0.02) as compared to 726 ± 39 and 627 ± 31 after testosterone and estradiol treatment. Reverse-phase high-performance liquid chromatography was performed on extracts of the MBH from individual castrated rats with and without DHT treatment in order to characterize the β-EP immunoactivity. The elution profile of the β-EP immunoactivity was very similar in both cases. Thus although DHT treatment is associated with a fall in the concentration of β-EP in the MBH, there does not appear to be a change in the processing of β-EP in the MBH. These results indicate that treatment with either testosterone or estradiol produces significant reductions in the hypothalamic concentrations of β-EP, CLIP, and α-MSH. The effectiveness of DHT in this respect demonstrates that there are independent androgen actions